Peptides having immunomodulatory properties

ABSTRACT

The present disclosure provides novel peptides that having immunomodulatory activities in vitro and in vivo. The peptides can include a particular striapathic region of alternating hydrophilic and hydrophobic modules that can adopt an amphipathic conformation under physiological conditions. This disclosure provides peptides that can specifically bind to key functional regions on one or more signaling proteins, particularly pro-inflammatory cytokines, macrophage inhibition proteins, and histone regulation proteins. This disclosure includes peptides that are sufficiently stable in the circulation to allow for intravenous administration. Pharmaceutical compositions including the subject peptides are also provided. The subject peptides find use in methods of modulating macrophage activity. In some cases, the peptide is a CD206-binding agent. Also provided are methods of treating a subject for a condition associated with chronic inflammation using the peptides and compositions of this disclosure.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/116,619 filed Aug. 29, 2018, which application is incorporated hereinby reference in its entirety.

INTRODUCTION

Acute inflammation is the initial response of a tissue to harmfulstimuli. It involves a complex, highly regulated process that beginswhen cells present in the injured tissue, including macrophages,dendritic cells, histiocytes, Kupffer cells, and mastocytes, sensemolecules associated with the injury and become activated. Uponactivation, these cells release inflammatory mediators, such asvasodilators. The vasodilators induce increased blood flow andpermeability of the blood vessels in the vicinity of the injury. This,in turn, results in the increased movement of plasma and leukocytes(including neutrophils and macrophages) from the blood into the injuredtissue. Because inflammatory mediators are, in general, rapidlydegraded, acute inflammation requires constant stimulation in order tobe sustained. As a result, acute inflammation ends once the harmfulstimulus is removed.

Various agents, including but not limited to bacteria, viruses, physicalinjury, chemical injury, cancer, chemotherapy, and radiation therapy,can, depending on the specific agent and the genetic makeup of theanimal exposed to it, cause prolonged and excessive inflammation. Suchinflammation, known as chronic inflammation, is believed to be acontributing factor to many widespread and debilitating diseases,including heart disease, cancer, respiratory disease, stroke,neurological diseases such as Alzheimer's disease, diabetes, and kidneydisease. The result of chronic inflammation is the destruction of normaltissue and its replacement with collagen-rich connective tissue.Collagen-rich connective tissue, also known as scar tissue, exhibitsdiminished tissue function as compared to normal tissue. Persistent andprolonged formation of scar tissue, in turn, leads to fibrosis. Fibrosisis among the common symptoms of diseases affecting the lungs, skin,liver, heart, and bone marrow, and is a critical factor in diseases suchas idiopathic pulmonary fibrosis, scleroderma, keloids, liver cirrhosis,myocardial fibrosis, diabetic kidney disease, myelodysplastic syndrome,and other disorders.

Studies of chronic inflammation and fibrosis have indicated that,regardless of the activating agent and the tissue affected, a commonnetwork of signaling proteins tend to function together to establish thepro-inflammatory state. This network of signaling proteins includes anumber of different cytokines, cytokine receptors, transcriptionfactors, and micro RNAs, including TGFβ, TGFβRII, and miRNA19b.Therapeutic agents that reduce inflammation without harmful side effectsare therefore of great interest.

SUMMARY

Novel peptides that have immunomodulatory activities in vitro and invivo are provided. The peptides can include a particular striapathicregion of alternating hydrophilic and hydrophobic modules that can adoptan amphipathic conformation under physiological conditions. The peptidescan specifically bind to key functional regions on one or more signalingproteins, particularly pro-inflammatory cytokines, macrophage inhibitionproteins, and/or histone regulation proteins. This disclosure includespeptides that are sufficiently stable in the circulation in vivo afteradministration to a subject. Pharmaceutical compositions including thesubject peptides are also provided.

The subject peptides find use in methods of modulating macrophageactivity. In some cases, the peptide is a CD206-binding agent. Alsoprovided are methods of treating a subject for a condition associatedwith chronic inflammation using the peptides and compositions of thisdisclosure.

These and other features and advantages of the compositions and methodsof the invention will be set forth or will become more fully apparent inthe description that follows and in the appended claims. For example,suitable immunomodulatory polypeptides may be identified by use of theformula and sequences described herein. Furthermore, features andadvantages of the described compositions and methods may be learned bypracticing the methods or will be obvious from the description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of results of reduction in bleomycin-induced lungfibrosis in a mouse model for lung fibrosis. The fibrosis measurementsare Ashcroft scores following trichrome staining. Collagen scores arequantitative measurements following hydroxyproline staining. Furtherdetails are provided in the experimental section below.

FIG. 2 demonstrates that exemplary peptides of interest synergize with aPD-1 Checkpoint Inhibitor to reduce tumor volume in a mouse tumorinhibition model. Further details are provided in the experimentalsection below.

FIG. 3 demonstrates that exemplary peptides RP832C and RP837 reduceviability of macrophages in samples from human scleroderma patients.Macrophage samples were assessed after 96 hours incubation with variousconcentrations of the peptides.

FIG. 4A-4B show the selective effect of exemplary peptides RP832C andRP837 on macrophage samples from scleroderma patients with higharginase:IFNg (interferon-gamma) ratio (FIG. 4B) versus samples fromhealthy controls with low arginase:IFNg ratio (FIG. 4A).

DETAILED DESCRIPTION

The following description supplies specific details in order to providea thorough understanding of the present invention. That said, to avoidobscuring aspects of the described immunomodulatory peptides and relatedmethods of treating a subject, well-known structures, materials,processes, techniques, and operations are not shown or described indetail. Additionally, the skilled artisan will understand that thedescribed immunomodulatory peptides and related methods of treating asubject can be implemented and used without employing these specificdetails. Indeed, the described immunomodulatory peptides and methods canbe placed into practice by modifying the illustrated peptides,compositions, kits and methods, and can be used in conjunction withother methods, treatments, apparatuses, and techniques usedconventionally.

Immunomodulatory Polypeptides

As summarized above, the present disclosure provides immune-modulatorypeptides, particularly peptides that have immunosuppressive properties,and methods of administering such immune-modulatory peptides to asubject, particularly a subject suffering from a medical conditionassociated with persistent or chronic inflammation or at risk ofdeveloping such a medical condition. The terms “immune-modulatory” and“immunomodulatory” are used interchangeably herein. In some cases, animmunomodulatory peptide described herein can be referred to as ananti-inflammatory peptide and vice versa. In certain instances, theimmunomodulatory peptide (e.g., as described herein) is ananti-inflammatory peptide, e.g., the peptide has at least oneanti-inflammatory property.

Certain aspects of immunomodulatory polypeptides of interest which maybe applied to, or adapted for use with, the peptides of the presentdisclosure are described by Jaynes et al. in WO2016/061133, thedisclosure of which is herein incorporated by reference in its entirety.

The terms “peptide” and “polypeptide” are used synonymously herein torefer to polymers constructed from amino acid residues. The term “aminoacid residue,” as used herein, refers to any naturally occurring aminoacid, non-naturally occurring amino acid, or amino acid mimetic (such asa peptoid monomer). An amino acid residue can be in an L- or D-form.

This disclosure includes immunomodulatory peptides having a striapathicregion that comprises at least 25% of the length of the polypeptide andat least one immunomodulatory property. The term “striapathic region,”refers to a region or portion of a peptide sequence that is composed ofa sequence of alternating hydrophobic and hydrophilic modules. A“hydrophobic module” is a peptide sequence consisting of one to five(e.g., 1 to 3 or 1 to 2) hydrophobic amino acid residues, e.g., 1, 2, 3,4 or 5 hydrophobic amino acid residues. A “hydrophilic module” is apeptide sequence consisting of one to five (e.g., 1 to 3 or 1 to 2)hydrophilic amino acid residues, e.g., 1, 2, 3, 4 or 5 hydrophilic aminoacid residues.

A striapathic region can thus be represented by the formulae(X₁₋₅J₁₋₅)_(n) or (J₁₋₅X₁₋₅)_(n), where each X signifies a hydrophilicamino acid residue, each J signifies a hydrophobic amino acid residue,and each n is an integer from 1 to 10, such as 2 to 10, 2 to 8, 3 to 8,4 to 8, or 5 to 10. As described in further detail below, aspects of thepresent disclosure include immunomodulatory peptides having astriapathic region having a specific degree of cationic charge.Immunomodulatory peptides of this disclosure can include an striapathicregion having a cationic surface. In certain embodiments, thestriapathic region has a cationic charge (i.e., charge >0, e.g., +1, +2,+3, +4, +5, +6 or more). In certain embodiments, the immunomodulatorypeptide includes a tail region (e.g., a hydrophobic tail sequence). Incertain embodiments, an immunomodulatory peptide includes two or morestriapathic regions. In such embodiments, two amphipathic regions of thepeptide are in the form of a dimer, where the two amphipathic regionscan have the same or different amino acid sequences (i.e., be ahomodimer or a heterodimer). In certain embodiments, the two (or more)striapathic regions are connected via a linker or linking region. Thelinker can be a contiguous (or in-line) amino acid sequence or anon-amino acid moiety as desired.

Hydrophobic amino acid residues are characterized by a sidechain groupthat has predominantly non-polar chemical or physical properties, e.g.,in an environment in which a peptide finds use, e.g., physiologicalconditions. Such hydrophobic amino acid residues can be naturallyoccurring or non-naturally occurring. A hydrophobic amino acid residuecan be a mimetic of a naturally occurring amino acid that ischaracterized by a sidechain group that has predominantly non-polarchemical or physical properties. Conversely, hydrophilic amino acidresidues are characterized by a sidechain group that is predominantlypolar (e.g., charged or neutral hydrophilic), e.g., in an environment inwhich a peptide finds use, e.g., physiological conditions. Suchhydrophilic amino acid residues can be naturally occurring ornon-naturally occurring. A hydrophilic amino acid residues can be amimetic of a naturally occurring amino acid characterized by a sidechaingroup that is predominantly hydrophilic (charged or neutral polar).Examples of hydrophilic and hydrophobic amino acid residues are shown inTable 1, below. Suitable non-naturally occurring amino acid residues andamino acid mimetics are known in the art. See, e.g., Liang et al.(2013), “An Index for Characterization of Natural and Non-Natural AminoAcids for Peptidomimetics,” PLoS ONE 8(7):e67844.

Although most amino acid residues can be considered as eitherhydrophobic or hydrophilic, a few, depending on their context, canbehave as either hydrophobic or hydrophilic. For example, due to theirrelatively weak non-polar characteristics, glycine, proline, serineand/or cysteine can sometimes function as hydrophilic amino acidresidues. Conversely, due to their bulky, slightly hydrophobic sidechains, histidine and arginine can sometimes function as hydrophobicamino acid residues.

TABLE 1 Hydrophobic and Hydrophilic Amino Acid Residues HydrophilicHydrophobic Residues (X) Residues (J) Arginine Tryptophan HistidinePhenylalanine Lysine Tyrosine Aspartic Acid Isoleucine Glutamic AcidLeucine Asparagine Valine Glutamine Methionine Pyrrolysine CysteineOrnithine Threonine Serine Alanine Proline Glycine SelenocysteineN-formylmethionine Norleucine Norvaline

The term “anti-inflammatory property,” as used herein, refers to anyproperty of a polypeptide that can be evaluated in silico, in vitro,and/or in vivo, that reduces or inhibits, or would be expected to reduceor inhibit, a pro-inflammatory signal mediated by a protein targetand/or reduces or inhibits inflammation in a subject. The term“immunomodulatory property,” as used herein, refers to any property of apolypeptide that can be evaluated in silico, in vitro, and/or in vivo,that modulates, or would be expected to modulate, expression orsecretion of one or more cytokines involved in autoimmunity and/orimmune responses to infectious agents, or by modulating one or morecomponents of a cytokine signalling pathway.

Selected Immunomodulatory Peptides of Interest

The exemplary immunomodulatory peptide sequences described herein aremerely examples and are not the only immunomodulatory polypeptidesprovided herein. Indeed, fragments and variants of the sequences of thedisclosed peptides are also within the scope of the present disclosure.

The present disclosure provides immunomodulatory polypeptides, sometimesreferred to as “RP peptides,” that satisfy one or more of the structuralformulae described below. The present disclosure also providesimmunomodulatory polypeptides that share a minimum degree of homologywith any of the exemplary RP peptides disclosed herein, or variantthereof, or a fragment thereof. Thus, a peptide or polypeptide of thepresent disclosure is an immunomodulatory peptide that satisfies one ofthe formulae described herein or shares a minimum degree of homologywith any of the exemplary RP peptides disclosed herein.

A “fragment” of the invention includes at least 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous aminoacid residues of a peptide disclosed herein (or up to one less than thenumber of amino acid residues in the subject peptide) and retains atleast one immunomodulatory property of the subject peptide. Thus,fragments of the invention include peptides that are missing one, two,three, four, or more amino acids from the N-terminus and/or theC-terminus relative to a parent immunomodulatory peptide disclosedherein.

A “variant” of the invention is a polypeptide that is substantiallysimilar to a polypeptide disclosed herein and retains at least oneimmunomodulatory property of the subject polypeptide. Variants caninclude deletions (i.e., truncations) of one or more amino acid residuesat the N-terminus or the C-terminus of a subject polypeptide disclosedherein; deletion and/or addition of one or more amino acid residues atone or more internal sites in the subject polypeptide disclosed herein;and/or substitution of one or more amino acid residues (e.g., one, two,three, or even more) at one or more positions in the subject polypeptidedisclosed herein. For subject polypeptides that are 12 amino acidresidues in length or shorter, variant polypeptides can include three orfewer (e.g., three, two, one, or none) deleted amino acid residues,whether located internally, at the N-terminal end, and/or at theC-terminal end.

Accordingly, the invention further provides immunomodulatorypolypeptides that are at least 50% identical (i.e., at least 50%sequence identity) (e.g., at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95% or more) to any one of theimmunomodulatory polypeptides disclosed in Tables disclosed herein(e.g., Table 3) and still retain at least one immunomodulatory property.Sequence identity is based on a comparison of two peptide sequences orfragments thereof of the same or similar length.

As such, in certain embodiments, this disclosure provides polypeptidesthat include an amino acid sequence having from 1 to 10 amino aciddifferences (e.g., 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 orfewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino aciddifference) to any one of the polypeptides disclosed herein and stillretain at least one immunomodulatory property. An “amino aciddifference” as used herein includes: an amino acid substitution, anamino acid insertion, a terminal amino acid addition, an amino aciddeletion, a terminal amino acid truncation, or any combination thereof.The differences between the striapathic region of a homologousimmunomodulatory polypeptide and any one of the immunomodulatorypolypeptides of Table 3 can include deletions, additions, and/orsubstitutions of amino acid residues, as discussed herein. Substitutedamino acid residues can be unrelated to the amino acid residue beingreplaced (e.g., unrelated in terms or hydrophobicity/hydrophilicity,size, charge, polarity, etc.), or the substituted amino acid residuescan constitute similar, conservative, or highly conservative amino acidsubstitutions. As used herein, “similar,” “conservative,” and “highlyconservative” amino acid substitutions are defined as shown in Table 2,below. The determination of whether an amino acid residue substitutionis similar, conservative, or highly conservative is based exclusively onthe side chain of the amino acid residue and not the peptide backbone,which may be modified to increase peptide stability, as discussed below.

TABLE 2 Classification of Amino Acid Substitutions Highly Amino AcidSimilar Conservative Conservative in Subject Amino Acid Amino Acid AminoAcid Polypeptide Substitutions Substitutions Substitutions Glycine (G)A, S, N A n/a Alanine (A) S, G, T, V, C, P, Q S, G, T S Serine (S) T, A,N, G, Q T, A, N T, A Threonine (T) S, A, V, N, M S, A, V, N S Cysteine(C) A, S, T, V, I A n/a Proline (P) A, S, T, K A n/a Methionine (M) L,I, V, F L, I, V L, I Valine (V) I, L, M, T, A I, L, M I Leucine (L) M,I, V, F, T, A M, I, V, F M, I Isoleucine (I) V, L, M, F, T, C V, L, M, FV, L, M Phenylalanine (F) W, Y, L, M, I, V W, L n/a Tyrosine (Y) F, W,H, L, I F, W F Tryptophan (W) F, L, V F n/a Asparagine (N) Q Q QGlutamine (Q) N N N Aspartic Acid (D) E E E Glutamic Acid (E) D D DHistidine (H) R, K R, K R, K Lysine (K) R, H, O R, H, O R, O Arginine(R) K, H, O K, H, O K, O Ornithine (O) R, H, K R, H, K K, R

Particular immunomodulatory peptide of interest, and fragments andvariants thereof which find us in the subject pharmaceuticalcompositions and methods are now described in greater detail. In certaincases, the subject immunomodulatory peptides have macrophage modulatingactivity.

The “length” of a polypeptide is the number of amino acid residueslinked end-to-end that constitute the polypeptide, excluding anynon-peptide linkers and/or modifications that the polypeptide maycontain. In some embodiments, the peptide is of 5 to 30 amino acidresidues (e.g., 5 to 25, 10 to 20 or 5 to 18, 5 to 12 or 5 to 10, or 6to 30, 6 to 25, 6 to 20, 6 to 18, 6 to 12, 6 to 10 or 7 to 12, or 7 to10 amino acid residues) in length, and comprises a striapathic region ofalternating hydrophilic and hydrophobic modules that adopts anamphipathic conformation under physiological conditions (e.g., asdescribed herein). In some embodiments, the peptide is of 5 to 12 aminoacid residues (e.g., 6, 7, 8, 9 or 10 amino acid residues) in length,and comprises a striapathic region of alternating hydrophilic andhydrophobic modules that adopts an amphipathic conformation underphysiological conditions. In certain instances, a striapathic region ofthe peptide is of 5 to 18 amino acid residues in length (e.g., 6 to 18,6 to 14, 6 to 12, 7 to 12, or 5, 6, 7, 8, 9, 10, 11 or 12 amino acids inlength), wherein the peptide is optionally further modified (e.g., asdescribed herein). The striapathic region can comprise: 2 or more (e.g.,3 or more or 4 or more) hydrophobic modules; and one or more (e.g., 2 ormore, 3 or more, or 4 or more) hydrophilic modules (e.g., eachcomprising at least one cationic residue). In some embodiments, thesubject immunomodulatory peptides (e.g., as described herein) areCD206-binding peptides. In some instances, the striapathic region of thepeptide has a length of 6 to 12 amino acid residues, such as 7 to 12. Insome instances, the striapathic region of the peptide has a length of 6to 10 amino acid residues.

The hydrophobic modules can consist of any convenient residues. Incertain instances, the hydrophobic modules include amino acid residuesselected from phenylalanine, tryptophan, alanine, valine, and glycine.The striapathic region can include 1, 2 or more cationic amino acidresidues in total, such as 3 or more, 4 or more, 5 or more, 6 or more,or even more. The immunomodulatory peptide can comprise 2, 3 or morehydrophilic modules that consist of any convenient residues. In someinstances, the hydrophilic modules include amino acid residues selectedfrom lysine, arginine, histidine, aspartic acid, glutamic acid,asparagine and glutamine.

In the formula described herein, J(N) is used to refer to a particularhydrophobic module, where N is indicates a position within the linearformula. Similarly, X(N) is used to refer to a particular hydrophilicmodule, where N is indicates a position within the linear formula.

In the formula described herein, J_((nx)) is used to refer to aparticular hydrophobic amino acid residue, where n indicates whichmodule the residue is located in and x indicates its position within themodule. Similarly, X_((nx)) is used to refer to a particular hydrophilicamino acid residue, where n indicates which module the residue islocated in and x indicates its position within the module.

In certain instances of the immunomodulatory peptide, the striapathicregion comprises hydrophobic and hydrophilic modules having thefollowing formula:[J1]-[X1]-[12]  (formula 1).

In some embodiments of the immunomodulatory peptide, the striapathicregion comprises the following formula of hydrophilic and hydrophobicmodules:[J1]-[X1]-[12]-[X2]  (formula 2)

In some embodiments of the immunomodulatory peptide, the striapathicregion comprises the following formula of hydrophilic and hydrophobicmodules:[X1]-[11]-[X2]-[12]  (formula 3).

In some embodiments of the immunomodulatory peptide, the striapathicregion comprises the following formula of hydrophobic and hydrophilicmodules:[J1]-[X1]-[J2]-[X2]-[J3]  (formula 4).

In certain embodiments, the striapathic region comprises three or morehydrophilic modules and three or more hydrophobic modules and comprisesone of the following formulae:[J1]-[X1]-[J2]-[X2]-[J3]-[X3]  (formula 5)[J1]-[X1]-[J2]-[X2]-[J3]-[X3]-[J4]  formula 6).

In certain embodiments, the striapathic region comprises three or morehydrophilic modules and three or more hydrophobic modules and comprisesone of the following formulae:[X1]-[J1]-[X2]-[J2]-[X3]-[J3]  (formula 7).

In some cases of formula 1, the striapathic region has a sequencedefined by one of the formulae:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)]  (formula 1A); and[J_(2b)J_(2a)]-[X_(1b)X_(1a)]-[J_(1b)J_(1a)]  (formula 1B);wherein:

J_(1a), J_(1b), J_(2a) and J_(2b) are each independently selected from ahydrophobic amino acid residue (e.g., phenylalanine, tryptophan andvaline); and

X_(1a) and X_(1b) are each independently selected from a hydrophilicamino acid residue (e.g., lysine or arginine).

In some instances of formula 1A, the peptide includes the sequenceFWKRFV (RP837N) (SEQ ID NO: 5), or a fragment or variant thereof (e.g.,a variant including one substitution).

In some embodiments of formula 2, the striapathic region has a sequencedefined by the formula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)]-[X_(2a)]  (formula 2A);wherein:

J_(1a), J_(1b), and J_(2a) are each independently selected from ahydrophobic amino acid residue (e.g., phenylalanine, tryptophan orvaline); and

X_(1a), X_(1b) and X_(2a) are each independently selected from ahydrophilic amino acid residue (e.g., lysine or arginine).

In some instances of formula 2A, the peptide includes the sequenceFVRKWR (RP837C¹) (SEQ ID NO: 6), or a fragment or variant thereof (e.g.,a variant including one substitution).

In some embodiments of formula 3, the striapathic region has a sequencedefined by the formula:[X_(1a)X_(1b)]-[J_(1a)J_(1b)J_(1c)J_(1d)]-[X_(2a)X_(2b)]-[J_(2a)J_(2b)]  (formula3A);wherein:

J_(1a), J_(1b), J_(1c), J_(1d), J_(2a) and J_(2b) are each independentlyselected from a hydrophobic amino acid residue (e.g., leucine, serine,alanine or phenylalanine); and

X_(1a), X_(1b), X_(2a) and X_(2b) are each independently selected from ahydrophilic amino acid residue (e.g., glutamic acid, aspartic acid,lysine, asparagine or arginine).

In some embodiments of formula 3A, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 25) EX_(1b)LSAFX_(2a)NJ_(2a)J_(2b);wherein:

J_(2a) and J_(2b) are each independently selected from alanine andphenylalanine; and

X_(1b) and X_(2a) are each independently selected from lysine andarginine.

In some instances of formula 3A, the peptide includes the sequenceEKLSAFRNFF (RP843) (SEQ ID NO: 9), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In certain instances of formula 4, the striapathic region has a sequencedefined by one of the formulae:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]-[J_(3a)J_(3b)]  (formula4A); and[J_(3a)J_(3b)]-[X_(2a)X_(2b)]-[J_(2b)J_(2a)]-[X_(1b)X_(1a)]-[J_(1b)J_(1a)]  (formula4B);wherein:

J_(1a), J_(1b), J_(2a), J_(2b), J_(3a) and J_(3b) are each independentlyselected from a hydrophobic amino acid residue (e.g., phenylalanine,tyrosine, isoleucine or leucine); and

X_(1a), X_(1b), X_(2a) and X_(2b) are each independently selected from ahydrophilic amino acid residue (e.g., lysine or arginine).

In some embodiments of formula 4A-4B, the striapathic region has asequence defined by the formula:

(SEQ ID NO: 26) LJ_(1b)KKIIKKJ_(3a)L

wherein J_(1b) and J_(1a) are independently phenylalanine, tyrosine orleucine (e.g., tyrosine or leucine).

In some instances of formula 4A-4B, the peptide includes the sequenceLYKKIIKKLL (RP846) (SEQ ID NO: 12), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some embodiments of formula 4, the striapathic region has a sequencedefined by one of the formulae:[J_(1a)J_(1b)J_(1c)]-[X_(1a)]-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]-[J_(3a)J_(3b)]  (formula4C);wherein:

J_(1a), J_(1b), J_(1c), J_(2a), J_(2b), J_(3a), and J_(3b) are eachindependently selected from a hydrophobic amino acid residue (e.g.,phenylalanine, tyrosine or proline); and

X_(1a), X_(2a) and X_(2b) are each independently selected from ahydrophilic amino acid residue (e.g., aspartic acid, lysine orarginine).

In some embodiments of formula 4C, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 27) FYPDJ_(2a)J_(2b)X_(2a)X_(2b)J_(3a)J_(3b)

wherein J_(2a), J_(2b), J_(3a), and J_(3b) are each independentlyphenylalanine or tyrosine (e.g., phenylalanine) X_(2a) and X_(2b) areeach independently lysine or arginine

In some instances of formula 4C, the peptide includes the sequenceFYPDFFKKFF (RP844) (SEQ ID NO: 10), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some embodiments of formula 4, the striapathic region has a sequencedefined by one of the formulae:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)]-[X_(2a)X_(2b)X_(2c)]-[J_(3a)J_(3b)]  (formula4D);wherein:

J_(1a), J_(1b), J_(2a), J_(3a) and J_(3b) are each independentlyselected from a hydrophobic amino acid residue (e.g., phenylalanine,serine, glycine or isoleucine); and

X_(1a), X_(1b), X_(2a), X_(2b) and X_(2c) are each independentlyselected from a hydrophilic amino acid residue (e.g., glutamic acid,aspartic acid, lysine or arginine).

In some embodiments of formula 4D, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 28) J_(1a)J_(1b)X_(1a)X_(1b)SKEKIGwherein:

J_(1a) and J_(1b) are each independently phenylalanine or tyrosine(e.g., phenylalanine); and X_(1a) and X_(1b) are each independentlylysine or arginine.

In some instances of formula 4D, the peptide includes the sequenceFFRKSKEKIG (RP853) (SEQ ID NO: 18), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In certain instances, the striapathic region has a sequence defined bythe formula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]-[J_(3a)]  (formula4E)wherein:

J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are each independentlyselected from phenylalanine, alanine and isoleucine; and

X_(1a), X_(1b), X_(2a), X_(2b) and X_(2c) are each independentlyselected from ornithine, lysine and arginine.

In certain instances, the striapathic region has a sequence defined bythe formula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]-[J_(3a)]-[X_(3a)]  (formula5A)wherein:

J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are each independentlyselected from phenylalanine, tryptophan, alanine and valine; and

X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are each independentlyselected from ornithine, lysine and arginine.

In some embodiments of formula 5A, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 29) J_(1a)J_(1b)OOJ_(2a)J_(2b)OOJ_(3a)Owherein J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are each independentlyselected from phenylalanine and alanine (e.g., each J1, J2 and J3 moduleincludes both phenylalanine and alanine).

In some embodiments of formula 5A, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 30) FAX_(1a)X_(1b)FAX_(2a)X_(2b)J_(3a)FX_(3a)wherein X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are each independentlyselected from ornithine, lysine and arginine.

In some instances of formula 5A, the peptide includes the sequenceFAOOFAOOFO (RP850) (SEQ ID NO: 19), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some embodiments of formula 5A, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 31) FWKX_(1b)FVX_(2a)KWX_(3a)wherein X_(1b), X_(2a) and X_(3a) are each independently lysine orarginine.

In some instances of formula 5A, the peptide includes the sequenceFWKRFVRKWR (RP837) (SEQ ID NO: 4) or FWKKFVKKWK (RP841) (SEQ ID NO: 7),or a fragment or variant thereof (e.g., a variant including one or twosubstitutions).

In some cases, the immunomodulatory peptide of formula 5A is notFFRKFAKRFK (RP183) (SEQ ID NO: 21) or FFKKFFKKFK (RP185) (SEQ ID NO:22).

In certain instances, the striapathic region has a sequence defined bythe formula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J₂]-[X_(2a)X_(2b)]-[J_(3a)]-[X_(3a)]  (formula5A)wherein:

J_(1a), J_(1b), J_(2a), J_(2b) and ba are each independently selectedfrom a hydrophobic amino acid residue (e.g., phenylalanine, tryptophan,alanine, valine, and glycine); and

X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are each independentlyselected from a hydrophilic amino acid residue (e.g., lysine, ornithine,arginine, histidine, aspartic acid, glutamic acid, asparagine orglutamine.

In some cases of formula 5A, J_(1a), J_(1b), J₂a, J_(2b) and J_(3a) areeach independently selected from phenylalanine, tryptophan, alanine andglycine; and X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are eachindependently selected from lysine and arginine. In certain instances offormula 5A, J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are eachindependently selected from phenylalanine, tryptophan, alanine andvaline; and X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are eachindependently selected from ornithine, lysine and arginine (e.g., Lys orArg). In some instances of formula 5A, J_(1a), J_(1b), J_(2a), J_(2b)and J_(3a) are each independently selected from phenylalanine andalanine; and X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are eachindependently selected from lysine and arginine. In certain cases offormula 5A, J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are eachphenylalanine; and X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are eachindependently selected from lysine and arginine. In some cases offormula 5A, J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are eachtryptophan; and X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are eachindependently selected from histidine, lysine and arginine. In someinstances of formula 5A, J_(1a), J_(2a) and J_(3a) are eachindependently selected from phenylalanine and tryptophan, J_(1b) isselected from tryptophan and alanine, J_(2b) is selected from valine,tryptophan and alanine and each of X_(1a), X_(1b), X_(2a), X_(2b) andX_(3a) are independently selected from ornithine, lysine, arginine orhistidine.

In some embodiments of formula 5A, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 32) WWX_(1a)HWWHX_(2b)WX_(3a)wherein X_(1a), X_(2b) and X_(3a) are each independently histidine,lysine or arginine.

In some instances of formula 5B, the peptide includes the sequenceWWHHWWHHWH (RP847) (SEQ ID NO: 13), WWRHWWHRWR (RP848) (SEQ ID NO: 14)or WWKHWWHKWK (RP849) (SEQ ID NO: 15), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some embodiments of formula 5, the striapathic region has a sequencedefined by the formula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)J_(2c)]-[X_(2b)]-[J_(3a)]-[X_(3a)]  (formula5B);

wherein:

J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are each independentlyselected from a hydrophobic amino acid residue (e.g., phenylalanine,alanine, threonine or leucine); and

X_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are each independentlyselected from a hydrophilic amino acid residue (e.g., histidine,aspartic acid, lysine or arginine).

In some embodiments of formula 5B, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 33) J_(1a)J_(1b)X_(1a)HJ_(2a)J_(2b)THLDwherein;

J_(1a), J_(1b), J_(2a) and J_(2b) are each independently selected fromphenylalanine and alanine; and

X_(1a) is independently selected from lysine and arginine.

In some instances of formula 5C, the peptide includes the sequenceFFRHFATHLD (RP845) (SEQ ID NO: 11), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some embodiments of formula 5, the striapathic region has a sequencedefined by one of the formulae:[J_(1a)]-[X_(1a)]-[J_(2a)J_(2b)J_(2c)]-[X_(2a)]-[J_(3a)J_(3b)]-[X_(3a)X_(3b)]  (formula5C);wherein:

J_(1a), J_(2a), J_(2b), J_(2c), J_(3a), and J_(3b) are eachindependently selected from a hydrophobic amino acid residue (e.g.,phenylalanine, tyrosine, leucine, glycine or isoleucine); and

X_(1a), X_(2a), X_(3a) and X_(3b) are each independently selected from ahydrophilic amino acid residue (e.g., glutamine, lysine or histidine).

In some embodiments of formula 5C, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 34) J_(1a)QJ_(2a)LGX_(2a)IIHHwherein:

J_(1a) and J_(2a) are each independently selected from phenylalanine,tyrosine and leucine; and

X_(2a) is lysine and arginine.

In some instances of formula 5C, the peptide includes the sequenceFQFLGKIIHH (RP852) (SEQ ID NO: 17), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some embodiments of formula 6, the striapathic region has a sequencedefined by the formula:[J_(1a)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)]-[X_(2a)]-[J_(3a)]-[X_(3a)X_(3b)]-[J_(4a)J_(4b)]  (formula6A);wherein:

J_(1a), J_(2a), J_(2b), J_(1a), J_(4a) and J_(4b) are each independentlyselected from a hydrophobic amino acid residue (e.g., phenylalanine,tryptophan, alanine, isoleucine, valine, and glycine); and

X_(1a), X_(1b), X_(2a), X_(3a) and X_(3b) are each independentlyselected from a hydrophilic amino acid residue (e.g., lysine, arginine,histidine, aspartic acid, glutamic acid, asparagine or glutamine).

In some embodiments of formula 6A, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 35) GX_(1a)X_(1b)GJ_(2b)X_(2a)GX_(3a)X_(3b)GJ_(4b)wherein:

J_(2b) and J_(4b) are each independently selected from phenylalanine,tryptophan, alanine, isoleucine and valine; and

X_(1a), X_(1b), X_(2a), X_(3a) and X_(3b) are each independentlyselected from lysine, arginine, histidine, aspartic acid, glutamic acid,asparagine and glutamine.

In some embodiments of formula 6A, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 36) GDX_(1b)GIX_(2a)GHX_(3b)GFwherein X_(1b), X_(2a) and X_(3b) are each independently selected fromlysine and arginine.

In some instances of formula 6A, the peptide includes the sequenceGDRGIKGHRGF (RP842) (SEQ ID NO: 8), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some embodiments of formula 7, the striapathic region has a sequencedefined by one of the formulae:[X_(1a)X_(1b)]-[J_(1a)]-[X_(2a)]-[J_(2a)]-[X_(3a)]-[J_(3a)J_(3b)J_(3c)]  (formula7A);wherein:

J_(1a), J_(2a), J_(3a), J_(3b), and J_(3c) are each independentlyselected from a hydrophobic amino acid residue (e.g., isoleucine,valine, leucine, serine or alanine); and

X_(1a), X_(1b), X_(2a) and X_(3a) are each independently selected from ahydrophilic amino acid residue (e.g., lysine or arginine).

In some embodiments of formula 7A, the striapathic region has a sequencedefined by the formula:

(SEQ ID NO: 37) X_(1a)X_(1b)IX_(2a)VX_(3a)LSAwherein X_(1a), X_(1b), X_(2a) and X_(3a) are each independentlyselected from lysine and arginine.

In some instances of formula 7A, the peptide includes the sequenceKKIRVRLSA (RP851) (SEQ ID NO: 16), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

Multimeric Peptides

This disclosure includes a multimer (e.g., a dimer) of two or moreimmunomodulatory peptides (e.g., as described herein) connected via abranced or liner linker. Aspects of the present disclosure includedimers of any of the subject immunomodulatory polypeptides. The dimerscan be homodimers or heterodimers. Any two immunomodulatory polypeptidescan be connected via a linker. Any convenient linker can be utilized.Linkers that may be employed include, but are not limited to, covalentbonds, peptide linkers (e.g., a glycine containing linker or a Gly andSer containing linker), C1-C12 linkers having terminal amino and orcarboxylic acid groups, or polymer linkers (e.g., a PEG or modifiedPEG). The dimers can include a linker that connects the C-terminal of afirst polypeptide with the N-terminal of a second polypeptide. Incertain cases, the two polypeptides can be linked via the C-terminals.In certain instances, the two polypeptides can be linked via theN-terminals.

The present disclosure further includes any two immunomodulatorypolypeptides which have been linked together. The linkage can be formedby a peptide linker, such as a Gly-Gly-Gly (GGG), Gly-Gly-Gly-Arg (GGGR;SEQ ID NO: 40), Gly-Pro-Gly (GPG), or Gly-Pro-Gly-Arg (GPGR; SEQ ID NO:41) sequence, that links the C-terminal end of a first immunomodulatorypolypeptide to the N-terminal end of a second immunomodulatorypolypeptide. Alternatively, the linkage can be a peptoid linker (e.g., apoly N-substituted version of any of the foregoing peptide linkers), apolymer containing g-amino acids (e.g., corresponding to any of theforegoing peptide linkers), or a non-peptide, chemical linker. Thelinked immunomodulatory polypeptides can be any of the polypeptidesdisclosed herein (e.g., in Table 3), and can include the samepolypeptide being linked to form a homodimer or different polypeptidesbeing linked to form a heterodimer. Techniques for linking peptides viapeptide and non-peptide linkers are well known in the art, and theinventive polypeptide combinations are intended to encompass all suchlinkages.

Any two striapathic region-containing peptides (e.g., as describedherein) can be linked. The two regions of a dimeric peptide can behomodimeric or heterodimeric with respect to each other. By homodimericis meant the two peptide regions of the dimeric peptide have the same Nto C sequence or a reversed C to N sequence thereof. The subjectimmunomodulatory polypeptides described herein can be linked in anyconvenient configuration to produce a multimer. In certain instances,the multimer includes 3 or more immunomodulatory polypeptides (e.g., asdescribed herein), where the polypeptides can be arranged in a linear orbranched fashion. A linear multimer of immunomodulatory polypeptides caninclude head to tail arrangement of linked peptides, linked via acovalent bond or an optional linker (e.g., a peptidic linker). In someinstances, a linear multimer can be referred to as an oligomer, e.g., apolypeptide chain that includes sequence segments of an immunomodulatorypolypeptides (e.g., as described herein). Alternatively, theimmunomodulatory polypeptides of a linear multimer can be linked via ahead to head (e.g., N-terminal to N-terminal linked) and/or tail to tail(e.g., C-terminal to C-terminal linked) configurations. In branchedmultimers the immunomodulatory polypeptides can be linked via anyconvenient branched linker, e.g., a group that includes three functionalgroups for attached to amino acid residues, such as a lysine amino acid.In some cases, the multimer is a dimer.

In certain cases the immunomodulatory peptide dimer has the formula:Z¹-T-Z²wherein:

T is a linker, e.g., a peptide linker;

Z¹ is a first polypeptide or region of 3-10 (e.g., 4-10, 5-10, or 3-6 or3, 4, 5, or 6) amino acid residues consisting of a mixture ofhydrophilic amino acid resides and hydrophobic amino acid residues (e.g.as described herein); and

Z² is a second polypeptide or region of 3-10 (e.g., 4-10, 5-10, or 3-6or 3, 4, 5, or 6) amino acid residues consisting of a mixture ofhydrophilic amino acid resides and hydrophobic amino acid residues (e.g.as described herein).

In certain cases of the dimer, the hydrophilic modules consist of aminoacid residues selected from lysine and arginine; and the hydrophobicmodules consist of amino acid residues selected from phenylalanine andtryptophan. In certain instances the first and second polypeptides (Z¹and Z²) comprise four amino acid residues. In certain cases each of Z¹and Z² comprises four amino acid residues, wherein two amino acidresidues are hydrophilic residues (e.g. as described herein) and theremaining two amino acid resides are hydrophobic residues (e.g. asdescribed herein).

In certain embodiments, the dimer has one of the following formulae:[X1]-[J1]-T-[J1]-[X1]  (formula 8);[J1]-[X1]-T-[X1]-[J1]  (formula 9);[X1]-[J1]-T-[J2]-[X2]  (formula 10);[J1]-[X1]-T-[X2]-[J2]  (formula 11);wherein T is the linker (e.g., peptide linker).

In some cases of formula 8 and 9, the dimer has a sequence defined byone of the following formulae:[X_(1a)X_(1b)]-[J_(1a)J_(1b)]-T-[J_(1b)J_(1a)]-[X_(1b)X_(1a)]  (formula8A); or[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-T-[X_(1b)X_(1a)]-[J_(1b)J_(1a)]  (formula9A);wherein:

T is the peptide linker (e.g., a polyglycine linker);

J_(1a) and J_(1b) are each independently selected from a hydrophobicamino acid residue (e.g. tryptophan or phenylalanine); and

X_(1a) and X_(1b) are each independently selected from a hydrophilicamino acid residue (e.g., asparagine or arginine). In certain instancesof formula 8A and 9A, T is a peptide linker consisting of one, two orthree glycine residues.

In some embodiments of formula 9A, the dimer has a sequence defined bythe formula:

(SEQ ID NO: 38) FW-[X_(1a)X_(1b)]-T-[X_(1b)X_(1a)]-WFwherein X_(1a), X_(1b) are each independently selected from lysine andarginine.

In some embodiments of formula 9A, the dimer has a sequence defined bythe formula:

(SEQ ID NO: 39) [J_(1a)J_(1b)]-KR-T-RK-[J_(1b)J_(1a)]wherein J_(1a) and J_(1b), are each independently selected fromtryptophan and phenylalanine.

In some instances of formula 7A, the peptide includes the sequenceFWKRGGRKWF (RP837A) (SEQ ID NO: 4), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some cases of formula 10 and 11, the dimer has a sequence defined byone of the following formulae:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-T-[X_(2a)X_(2b)]-[J_(2a)J_(2b)]  (formula10A)[X_(1a)X_(1b)]-[J_(1a)J_(1b)]-T-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]  (formula11A)wherein:

J_(1a), J_(1b), J_(2a) and J_(2b) are each independently selected from ahydrophobic amino acid residue (e.g. tryptophan or phenylalanine); and

X_(1a), X_(1b), X_(2a) and X_(2b) are are each independently selectedfrom a hydrophilic amino acid residue (e.g., asparagine or arginine). Incertain instances of formula 10A and 11A, T is a peptide linkerconsisting of one, two or three glycine residues.

In certain instances, the first and second polypeptides (Z¹ and Z²) ofthe dimer comprise one of the following formulae of hydrophilic andhydrophobic modules: [X1]-[J1]-[X2]-[J2] (formula 3); or[J1]-[X1]-[J2]-[X2] (formula 2).

In certain embodiments, the dimer has one of the following formulae:[X1]-[J1]-[X2]-[J2]-T-[J2]-[X2]-[J1]-[X1]  (formula 12);[J1]-[X1]-[J2]-[X2]-T-[X2]-[J2]-[X1]-[J1]  (formula 13);[X1]-[J1]-[X2]-[J2]-T-[J3]-[X3]-[J4]-[X4]  (formula 14);[J1]-[X1]-[J2]-[X2]-T-[X3]-[J3]-[X4]-[J4]  (formula 15);wherein T is the peptide linker.

In some instances of formula 12 and 13, the dimer has one of thefollowing formulae:[X_(1a)]-[J_(1a)]-[X_(2a)]-[J_(2a)]-T-[J_(2a)]-[X_(2a)]-[J_(1a)]-[X_(1a)]  (formula12A); and[J_(1a)]-[X_(1a)]-[J_(2a)]-[X_(2a)]-T-[X_(2a)]-[J_(2a)]-[X_(1a)]-[J_(1a)]  (formula13A);wherein:

T is the peptide linker (e.g., a polyglycine linker);

J_(1a) and J_(2a) are each independently selected from phenylalanine andtryptophan; and

X_(1a) and X_(2a) are each independently selected from lysine andarginine.

In some instances of formula 12A, the peptide includes the sequenceRWKFGGFKWR (RP832C) (SEQ ID NO: 1), or a fragment or variant thereof(e.g., a variant including one or two substitutions).

In some instances of formula 13A, the peptide includes the sequenceFKWRGGRWKF (RP837C) (SEQ ID NO: 3), or a fragment or variant thereof(e.g., a variant including one or two substitutions). In certainembodiments, an immunomodulatory peptide includes a tail region.

In some instances of formula 14 and 15, the dimer has one of thefollowing formulae:[X_(1a)]-[J_(1a)]-[X_(2a)]-[J_(2a)]-T-[J_(3a)]-[X_(3a)]-[J_(4a)]-[X_(4a)]  (formula14A); and[J_(1a)]-[X_(1a)]-[J_(2a)]-[X_(2a)]-T-[X_(3a)]-[J_(3a)]-[X_(4a)]-[J_(4b)]  (formula15A);wherein:

T is the peptide linker (e.g., a polyglycine linker);

J_(1a), J_(2a), J_(3a), and J_(4a) are each independently selected fromphenylalanine and tryptophan; and

X_(1a), X_(2a), X_(3a) and X_(4a) are each independently selected fromlysine and arginine.

Immunomodulatory peptides of interest include, but are not limited to,any one of the polypeptides of Table 3, a fragment thereof (e.g., asdescribed herein), or a variant thereof (e.g., as described herein).

TABLE 3 selected peptides of interest RP# SEQ ID NO: Sequence 832C  1RWKFGGFKWR 837   2 FWKRFVRKWR 837C  3 FKWRGGRWKF 837A  4 FWKRGGRKWF 837N 5 FWKRFV 837C¹  6 FVRKWR 841   7 FWKKFVKKWK 842   8 GDRGIKGHRGF 843   9EKLSAFRNFF 844  10 FYPDFFKKFF 845  11 FFRHFATHLD 846  12 LYKKIIKKLL 847 13 WWHHWWHHWH 848  14 WWRHWWHRWR 849  15 WWKHWWHKWK 851  16 KKIRVRLSA852  17 FQFLGKIIHH 853  18 FFRKSKEKIG 850  19 FAOOFAOOFO

In certain embodiments, the subject immunomodulatory polypeptideincludes a sequence selected from:

-   -   a) a sequence selected from the peptide sequences of Table 3;    -   b) a sequence having at least 75% sequence identity (e.g., at        least 80%, at least 85%, at least 90% or at least 95% sequence        identity) with the sequence defined in a); and    -   c) a sequence having one or two amino acid substitutions        relative to the sequence defined in a), wherein the one or two        amino acid substitutions are substitutions for amino acids        according to Table 2 (e.g., a similar amino acid substitution, a        conservative amino acid substitutions or a highly conservative        amino acid substitution).

In certain cases, the sequence set forth in a) is RP 832C. In certaincases, the sequence set forth in a) is RP 837. In certain cases, thesequence set forth in a) is RP 837C. In certain cases, the sequence setforth in a) is RP 837A. In certain cases, the sequence set forth in a)is RP 837N. In certain cases, the sequence set forth in a) is RP 837C¹.In certain cases, the sequence set forth in a) is RP 841. In certaincases, the sequence set forth in a) is RP 842. In certain cases, thesequence set forth in a) is RP 843. In certain cases, the sequence setforth in a) is RP 844. In certain cases, the sequence set forth in a) isRP 845. In certain cases, the sequence set forth in a) is RP 846. Incertain cases, the sequence set forth in a) is RP 847. In certain cases,the sequence set forth in a) is RP 848. In certain cases, the sequenceset forth in a) is RP 849. In certain cases, the sequence set forth ina) is RP 850. In certain cases, the sequence set forth in a) is RP 851.In certain cases, the sequence set forth in a) is RP 852. In certaincases, the sequence set forth in a) is RP 853.

In certain instances, the sequence set forth in b) has a sequence havingat least 80% sequence identity with the sequence defined in a). Incertain instances, the sequence set forth in b) has a sequence having atleast 85% sequence identity with the sequence defined in a). In certaininstances, the sequence set forth in b) has a sequence having at least90% sequence identity with the sequence defined in a). In certaininstances, the sequence set forth in b) has a sequence having at least95% sequence identity with the sequence defined in a).

In certain embodiments, the sequence set forth in c) has one or twoamino acid substitutions relative to the sequence defined in a), whereinthe one or two amino acid substitutions are similar amino acidsubstitutions according to Table 2. In certain embodiments, the sequenceset forth in c) has one or two amino acid substitutions relative to thesequence defined in a), wherein the one or two amino acid substitutionsare conservative amino acid substitutions according to Table 2. Incertain embodiments, the sequence set forth in c) has one or two aminoacid substitutions relative to the sequence defined in a), wherein theone or two amino acid substitutions are highly conservative amino acidsubstitutions according to Table 2. Any of the variations ofimmunomodulatory peptides described herein may be applied to the parentpeptides of Table 3.

Excluded Polypeptides

Compositions of the present disclosure optionally exclude polypeptidesdescribed in US Patent Application Nos. 2012/0270770 and 2003/0109452,and U.S. Pat. No. 6,559,281, the disclosures of which are hereinincorporated by reference in their entirety. Accordingly, one or morepolypeptides and/or uses of such polypeptides described in suchpublications can be excluded from the scope of the presently disclosedcomposition and/or methods. Moreover, any of the polypeptides disclosedin Tables 3-9 of WO2016/061133 by Jaynes et al., the disclosure of whichtables is herein incorporated by reference, can be optionally excludedfrom the compositions disclosed herein and/or methods of using suchcompounds. In some cases, any of the polypeptides disclosed in instantTable 4 can be optionally excluded from compositions disclosed hereinand/or methods of using such compounds.

In some cases, the immunomodulatory peptide of the formulae describedherein is NOT a polypeptide of Table 4.

TABLE 4 RP# SEQ ID NO: Sequence 182 20 KFRKAFKRFF 183 21 FFRKFAKRFK 18522 FFKKFFKKFK 186 23 KFKKFFKKFF 233 24 KFKKAFKKAFModified Polypeptides

Embodiments of the present disclosure include the modification of any ofthe immunomodulatory polypeptides of the present disclosure, by chemicalor genetic means. Examples of such modification include construction ofpeptides of partial or complete sequence with non-natural amino acidsand/or natural amino acids in L or D forms. For example, any of thepeptides disclosed herein and any variants thereof could be produced inan all-D form. Furthermore, polypeptides of the present disclosure canbe modified to contain carbohydrate or lipid moieties, such as sugars orfatty acids, covalently linked to the side chains or the N- or C-terminiof the amino acids. In addition, the polypeptides of the presentdisclosure can be modified to enhance solubility and/or half-life uponbeing administered. For example, polyethylene glycol (PEG) and relatedpolymers have been used to enhance solubility and the half-life ofprotein therapeutics in the blood. Accordingly, the polypeptides of thepresent disclosure can be modified by PEG polymers and the like.Polypeptides of the present disclosure can also be modified to containsulfur, phosphorous, halogens, metals, etc. And amino acid mimics can beused to produce polypeptides of the present disclosure (e.g., having astructure based on the Structural Algorithm or a structure similar toany of the immunomodulatory polypeptides disclosed herein). In certainembodiments, polypeptides of the present disclosure that include aminoacid mimics have enhanced properties, such as resistance to degradation.For example, polypeptides of the present disclosure can include one ormore (e.g., all) peptoid monomers.

Immunomodulatory polypeptides can be linked to another molecule via abiodegradable linkage, such as a disulfide bond. The disulfide bond canbe mediated by the sulfhydryl group of a cysteine residue found in theimmunomodulatory polypeptide and a sulfhydryl group in the othermolecule. The cysteine residue can be, e.g., located at either theC-terminal or N-terminal end of immunomodulatory polypeptide. Using adisulfide linkage of this sort, polypeptides of the present disclosurecan be conveniently linked to various types of useful molecules. Forexample, the linkage can be with another immunomodulatory polypeptide(which optionally includes a C-terminal or N-terminal cysteine residue),a fluorescent label (e.g., Dylight 350), a chemotherapeutic agent (e.g.,a taxol derivative formed by adding a sulfhydral group to an appropriatesite on the taxol ring structure, followed by oxidation with acysteine-containing peptide of the present disclosure), or the like.

Linked immunomodulatory polypeptides (e.g., homo- or heterodimers) canbind to a target molecule (e.g., a target protein, such as apro-inflammatory signaling protein) with a binding energy that isgreater than that of either monomer polypeptide alone. Thus, forexample, the energy of binding of linked immunomodulatory polypeptidesto an NF-kB Class II protein (e.g., RelB) can be at least −700 kcal/mol,and in certain embodiments at least −750, −800, −900, −1000, −1100,−1200, −1250, −1300, −1350, −1400, −1425, −1450, −1475, −1500, −1525,−1550, −1575, −1600 kcal/mol, or greater. The energy of binding can bedetermined, e.g., in silico, in vitro, or in vivo, using methodswell-known in the art (e.g., using the ClusPro™ algorithm).

In some instances, where the modified peptide is covalently linked to amolecule of interest, the resulting compound can be termed a peptideconjugate. Any convenient molecules of interest may be attached to thesubject immunomodulatory peptides. The molecule of interest may bepeptidic or non-peptidic, naturally occurring or synthetic. Molecules ofinterest suitable for use in conjunction with the subjectimmunomodulatory peptides include, but are not limited to, a proteindomain, a polypeptide, a peptide tag, a specific binding moiety (e.g.,an antibody or antibody fragment), a polymeric moiety such as apolyethylene glycol (PEG), a carbohydrate, a dextran or a polyacrylate,a linker, a moiety that imparts desirable drug-like properties such as ahalf-life extending moiety, a label and a solid support. In some cases,the molecule of interest may confer on the resulting modified peptidesenhanced and/or modified properties and functions including, but notlimited to, increased water solubility, ease of chemical synthesis,cost, bioconjugation site, stability, pI, aggregation, reducednon-specific binding and/or specific binding to a second target protein,e.g., as described herein.

In some embodiments of any one of the peptide sequences describedherein, the peptide sequence may be extended to include one or moreadditional residues at the N-terminal and/or C-terminal of the sequence,such as two or more, three or more, four or more, five or more, 6 ormore, or even more additional residues. Any convenient residues may beincluded at the N-terminal and/or C-terminal of the peptide to providefor a desirable property or group, such as increased solubility via awater soluble group, a linkage for dimerization or multimerization, alinkage for connecting to a label or a specific binding moiety.

In some cases, the subject modified peptide is described by formula:B-L-Mwhere B is an immunomodulatory peptide (e.g., as described herein); L isan optional linking group; and M is a molecule of interest, where L isattached to B at any convenient location (e.g., the N-terminal,C-terminal or via the sidechain of a residue not involved in binding tothe target).

The modified peptides may include one or more molecules of interest. Insome instances, the molecule of interest is covalently attached via thealpha-amino group of the N-terminal residue, or is covalently attachedto the alpha-carboxyl acid group of the C-terminal residue.

The molecules of interest may include a polypeptide or a protein domain.Polypeptides and protein domains of interest include, but are notlimited to: gD tags, c-Myc epitopes, FLAG tags, His tags, fluorescenceproteins (e.g., GFP), beta-galactosidase protein, GST, albumins,immunoglobulins, antibodies, Fc domains, or similar antibody-likefragments, leucine zipper motifs, a coiled coil domain, a hydrophobicregion, a hydrophilic region, a polypeptide comprising a free thiolwhich forms an intermolecular disulfide bond between two or moremultimerization domains, a “protuberance-into-cavity” domain,beta-lactoglobulin, or fragments thereof.

The molecules of interest may include a half-life extending moiety. Theterm “half-life extending moiety” refers to a pharmaceuticallyacceptable moiety, domain, or “vehicle” covalently linked or conjugatedto the subject compound, that prevents or mitigates in vivo proteolyticdegradation or other activity-diminishing chemical modification of thesubject compound, increases half-life or other pharmacokineticproperties (e.g., rate of absorption), reduces toxicity, improvessolubility, increases biological activity and/or target selectivity ofthe subject compound with respect to a target of interest, increasesmanufacturability, and/or reduces immunogenicity of the subjectcompound, compared to an unconjugated form of the subject compound.

In certain embodiments, the half-life extending moiety is a polypeptidethat binds a serum protein, such as an immunoglobulin (e.g., IgG) or aserum albumin (e.g., human serum albumin (HSA)). Polyethylene glycol isan example of a useful half-life extending moiety. Exemplary half-lifeextending moieties include a polyalkylene glycol moiety (e.g., PEG), aserum albumin or a fragment thereof, a transferrin receptor or atransferrin-binding portion thereof, and a moiety comprising a bindingsite for a polypeptide that enhances half-life in vivo, a copolymer ofethylene glycol, a copolymer of propylene glycol, acarboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, apoly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, apolyaminoacid (e.g., polylysine), a dextran n-vinyl pyrrolidone, a polyn-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxidepolymer, an ethylene oxide polymer, a polyoxyethylated polyol, apolyvinyl alcohol, a linear or branched glycosylated chain, a polysialicacid, a polyacetal, a lipid, a long chain fatty acid, a long chainhydrophobic aliphatic group, an immunoglobulin Fc domain (see, e.g.,U.S. Pat. No. 6,660,843), an albumin (e.g., human serum albumin; see,e.g., U.S. Pat. No. 6,926,898 and US 2005/0054051; U.S. Pat. No.6,887,470), a transthyretin (TTR; see, e.g., US 2003/0195154;2003/0191056), or a thyroxine-binding globulin (TBG).

In certain embodiments, the half-life extending moiety is a lipid. Incertain embodiments, the half-life extending moiety is a fatty acid. Anyconvenient lipids and fatty acids may be used in the subject modifiedcompounds. See e.g., Chae et al., “The fatty acid conjugated exendin-4analogs for type 2 antidiabetic therapeutics”, J. Control Release. 2010May 21; 144(1):10-6.

In certain embodiments, the immunomodulatory peptide is modified toinclude a specific binding moiety. The specific binding moiety is amoiety that is capable of specifically binding to a second moiety thatis complementary to it. In some cases, the specific binding moiety bindsto the complementary second moiety with an affinity of at least 10⁻⁷ M(e.g., as measured by a K_(D) of 100 nM or less, such as 30 nM or less,10 nM or less, 3 nM or less, 1 nM or less, 300 pM or less, or 100 pM oreven less). Complementary binding moiety pairs of specific bindingmoieties include, but are not limited to, a ligand or activator/promoterand a receptor, an antibody and an antigen, complementarypolynucleotides, complementary protein homo- or heterodimers, an aptamerand a small molecule, and a polyhistidine tag and nickel. The specificbinding pairs may include analogs, derivatives and fragments of theoriginal specific binding member. For example, an antibody directed to aprotein antigen may also recognize peptide fragments, chemicallysynthesized, labeled protein, derivatized protein, etc. so long as anepitope is present. Protein domains of interest that find use asspecific binding moieties include, but are not limited to, Fc domains,or similar antibody-like fragments, leucine zipper motifs, a coiled coildomain, a hydrophobic region, a hydrophilic region, a polypeptidecomprising a free thiol which forms an intermolecular disulfide bondbetween two or more multimerization domains, or a“protuberance-into-cavity” domain (see e.g., WO 94/10308; U.S. Pat. No.5,731,168, Lovejoy et al. (1993), Science 259: 1288-1293; Harbury et al.(1993), Science 262: 1401-05; Harbury et al. (1994), Nature 371:80-83;Hakansson et al. (1999), Structure 7: 255-64.

In certain embodiments, the peptide is a linked specific binding moietythat specifically binds a target protein. The linked specific bindingmoiety can be an antibody, an antibody fragment, a receptor activator,or an aptamer. The linked specific binding moiety can specifically bindany convenient target protein, e.g., a target protein that is desirableto target in conjunction with the subject methods of treatment. Targetproteins of interest include, but are not limited to, PDGF (e.g.,PDGF-B), VEGF-B, VEGF-C, VEGF-D, EGF, EGFR, Her2, PD-1, PD-L1, OX-40 andLAG3. In certain embodiments, the linked specific binding moiety is areceptor activator or ligand, e.g., a protein ligand associated with aninflammatory pathway, such as interleukin 13 (IL-13) or a molecule thatactivates is a member of the toll-like receptor (TLR) family, e.g.,TLR3. In certain instances, the linked specific binding moiety (e.g., aprotein, antibody, or antibody fragment) can be further linked to anadditional active agent (e.g., a chemotherapeutic agent, e.g., asdescribed herein).

An immunomodulatory polypeptide (e.g., as described herein) may beconjugated to an additional active agent to provide a conjugate of animmunomodulatory polypeptide. Once the subject peptides have beengenerated and/or fabricated and selected according to the teachingsherein they may be linked with, fused to, conjugated to (e.g.,covalently or non-covalently) or otherwise associated withpharmaceutically active or diagnostic moieties or biocompatiblemodifiers. The term “peptide conjugate” refers to any biologicallyactive or detectable molecule or drug associated with the disclosedimmunomodulatory peptide compound regardless of the method ofassociation. In this respect it will be understood that such conjugatesmay, in addition to the disclosed immunomodulatory peptides, comprisepeptides, polypeptides, proteins, prodrugs which are metabolized to anactive agent in vivo, polymers, nucleic acid molecules, small molecules,binding agents, mimetic agents, synthetic drugs, inorganic molecules,organic molecules and radioisotopes. Moreover, as indicated above theselected conjugate may be covalently or non-covalently associated with,or linked to, the subject peptide and exhibit various stoichiometricmolar ratios depending, at least in part, on the method used to effectthe conjugation.

In certain instances, the molecule of interest is a second active agent,e.g., an active agent or drug that finds use in conjunction with targetsof interest in the subject methods of treatment. In certain instances,the molecule of interest is a small molecule, a chemotherapeutic, anantibody, an antibody fragment, a bispecific antibody, an aptamer, or aL-protein. In some embodiments, the peptide is modified to include amoiety that is useful as a pharmaceutical (e.g., a protein, nucleicacid, organic small molecule, etc.). Exemplary pharmaceutical proteinsinclude, e.g., cytokines, antibodies, chemokines, growth factors,interleukins, cell-surface proteins, extracellular domains, cell surfacereceptors, cytotoxins, etc. Exemplary small molecule pharmaceuticalsinclude small molecule toxins or therapeutic agents. Any convenienttherapeutic or diagnostic agent (e.g., as described herein) can beconjugated to an immunomodulatory peptide. A variety of therapeuticagents including, but not limited to, anti-cancer agents,antiproliferative agents, cytotoxic agents and chemotherapeutic agentsare described below in the section entitled Combination Therapies, anyone of which can be adapted for use in the subject peptide conjugates.

In certain embodiments, the modified peptide may be conjugated to abispecific antibody, e.g., an engineered bispecific monoclonal antibodythat can simultaneously bind to two different types of antigen ofinterest.

In certain embodiments, the modified peptide may include a cellpenetrating peptide (e.g., tat). The cell penetrating peptide mayfacilitate cellular uptake of the molecule. Any convenient tagpolypeptides and their respective antibodies may be used. Examplesinclude poly-histidine (poly-his) or poly-histidine-glycine(poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5[Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptidesinclude the Flag-peptide [Hopp et al., BioTechnology 6:1204-1210(1988)]; the KT3 epitope peptide [Martin et al., Science 255:192-194(1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem.266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A. 87:6393-6397(1990)].

Those skilled in the art will appreciate that a number of differentreactions are available for the attachment or association of therapeuticor diagnostic moieties and/or linkers to the subject immunomodulatorypeptides. In certain embodiments, this may be accomplished by reactionof the amino acid residues of the peptide, e.g., as described herein,including the amino terminal, the C-terminal carboxylic acid, the aminegroups of lysine, the free carboxylic acid groups of glutamic andaspartic acid, the sulfhydryl groups of cysteine and the variousmoieties of the aromatic amino acids. One method of covalent attachmentis the carbodiimide reaction to link a carboxy (or amino) group of acompound to amino (or carboxy) groups of the subject peptide.Additionally, bifunctional agents such as dialdehydes or imidoestershave been used to link the amino group of a subject peptide to aminogroups of an antibody molecule. Also available for attachment of drugsto immunomodulatory peptides is maleimide—thiol conjugation chemistry,Click chemistry, e.g., between an azido and an alkynyl group, and thelike. Also available for attachment of drugs to peptides is the Schiffbase reaction. This method can involve the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatesand azlactones can also be used as coupling agents for covalentlyattaching drugs to binding agents.

It will be appreciated that several varieties or types of linker may beused to associate the disclosed immunomodulatory peptides withpharmaceutically active or diagnostic moieties or biocompatiblemodifiers. In some embodiments, the linker is cleavable underintracellular conditions, such that cleavage of the linker releases thedrug unit from the antibody in the intracellular environment. In certainembodiments, the linker unit is not cleavable. Bivalent linker reagentswhich are useful to attach two or more functional or biologically activemoieties, such as peptides, nucleic acids, drugs, toxins, antibodies,haptens, and reporter groups are known, and methods have been describedtheir resulting conjugates (Hermanson, G. T. (1996) BioconjugateTechniques; Academic Press: New York, p 234-242).

Compositions

Compositions of the present disclosure include an immunomodulatorypolypeptide that satisfies one of the structural formula describedherein. For example, the immunomodulatory polypeptide can have astriapathic region having a sequence that conforms with any one ofFormulas disclosed herein. Typically, the immunomodulatory polypeptideincluded in the compositions of the present disclosure will be asynthetic polypeptide (e.g., made by chemical synthesis and/or producedrecombinantly).

The compositions of the present disclosure can include a singleimmunomodulatory polypeptide, or combinations thereof. The compositionscan be substantially free of proteins and other polypeptides that do notsatisfy the structural algorithm disclosed herein. As used herein, theterm “substantially free of proteins and other polypeptides” means thatless than 5% of the protein content of the composition is made up ofproteins and other polypeptides that are not an immunomodulatorypolypeptide of the present disclosure. A composition that issubstantially free of non-immunomodulatory polypeptides of the presentdisclosure can have less than 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%,or less of proteins or other polypeptides that do not satisfy thestructural algorithm disclosed herein. Thus, the compositions can besubstantially free of blood proteins, such as serum albumin, globulins,fibrinogen, and clotting factors. Alternatively, the compositions can besubstantially free of globulins, fibrinogen, and clotting factors, butcan include purified or recombinantly produced serum albumin.

The compositions of the present disclosure in certain embodimentscontain an immunomodulatory polypeptide that is not naturally found in ahuman or other mammal or animal. However, compositions of the presentdisclosure can include an immunomodulatory polypeptide that is naturallyfound in a human or other mammal or animal, provided that thecomposition is substantially free of biological molecules (such asnon-immunomodulatory polypeptides, nucleic acids, lipids, carbohydrates,and metabolites) that are associated with the immunomodulatorypolypeptide in vivo or co-purify with the immunomodulatory polypeptide.As used herein, the term “substantially free of biological molecules”means that less than 5% of the dry weight of the composition is made upof biological molecules that are not immunomodulatory polypeptides. Acomposition that is substantially free of such biological molecules canhave less than 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less ofbiological molecules that are not immunomodulatory polypeptides. Thus,for example, the composition can be substantially free of biologicalmolecules that are abundant in the blood, such the proteins discussedabove, fatty acids, cholesterol, non-protein clotting factors,metabolites, and the like. In addition, the composition can besubstantially free of cells, including red blood cells, white bloodcells, and platelets, and cell fragments.

The compositions of the present disclosure can include at least 1 mg(e.g., at least 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400,500, 600, 700, 800, 900, 1000 mg, or more) of immunomodulatorypolypeptide. Thus, for example, the compositions can include an amountof immunomodulatory polypeptide equal to about 1 mg to about 1000 mg(e.g., about 5 mg to about 900 mg, about 5 mg to about 800 mg, about 5mg to about 700 mg, about 5 mg to about 600 mg, about 10 mg to about 500mg, about 10 mg to about 400 mg, about 10 mg to about 300 mg, about 10mg to about 250 mg, about 10 mg to about 200 mg, about 10 mg to about150 mg, about 10 mg to about 100 mg, about 50 mg to about 500 mg, about50 mg to about 400 mg, about 50 mg to about 300 mg, about 50 mg to about250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about50 mg to about 100 mg, about 75 mg to about 500 mg, about 75 mg to about400 mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg, about75 mg to about 200 mg, about 75 mg to about 150 mg, about 75 mg to about100 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg,about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mgto about 200 mg, or any other range containing two of the foregoingendpoints).

The compositions of the present disclosure can include a solution thatcontains at least 1 mg/ml (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/ml or more) of animmunomodulatory polypeptide. Thus, for example, the compositions caninclude a solution having an immunomodulatory polypeptide concentrationof about 1 mg/ml to about 1000 mg/ml (e.g., about 5 mg/ml to about 900mg/ml, about 5 mg/ml to about 800 mg/ml, about 5 mg/ml to about 700mg/ml, about 5 mg/ml to about 600 mg/ml, about 5 mg/ml to about 500mg/ml, about 10 mg/ml to about 500 mg/ml, about 10 mg/ml to about 400mg/ml, about 10 mg/ml to about 300 mg/ml, about 10 mg/ml to about 250mg/ml, about 10 mg/ml to about 200 mg/ml, about 10 mg/ml to about 150mg/ml, about 10 mg/ml to about 100 mg/ml, about 50 mg/ml to about 500mg/ml, about 50 mg/ml to about 400 mg/ml, about 50 mg/ml to about 300mg/ml, about 50 mg/ml to about 250 mg/ml, about 50 mg/ml to about 200mg/ml, about 50 mg/ml to about 150 mg/ml, about 50 mg/ml to about 100mg/ml, about 75 mg/ml to about 500 mg/ml, about 75 mg/ml to about 400mg/ml, about 75 mg/ml to about 300 mg/ml, about 75 mg/ml to about 250mg/ml, about 75 mg/ml to about 200 mg/ml, about 75 mg/ml to about 150mg/ml, about 75 mg/ml to about 100 mg/ml, about 100 mg/ml to about 500mg/ml, about 100 mg/ml to about 400 mg/ml, about 100 mg/ml to about 300mg/ml, about 100 mg/ml to about 250 mg/ml, about 100 mg/ml to about 200mg/ml, about 10 mg/ml to about 150 mg/ml, or any other range containingtwo of the foregoing endpoints).

The compositions of the present disclosure include pharmaceuticalcompositions. Such pharmaceutical compositions can comprise one or moreimmunomodulatory polypeptides and a pharmaceutically acceptable carrier.Pharmaceutical compositions can further include a protein other than animmunomodulatory polypeptide of the present disclosure and/or achemotherapeutic agent. The other protein can be a therapeutic agent,such as a therapeutic antibody. The therapeutic protein or antibody canhave immunomodulatory properties or other properties that theimmunomodulatory polypeptides of the present disclosure augment or areaugmented by. Alternatively, the other protein can be a carrier protein,such as serum albumin (e.g., HSA). The serum albumin (e.g., HAS, BSA,etc.) can be purified or recombinantly produced. By mixing theimmunomodulatory polypeptide(s) in the pharmaceutical composition withserum album, the immunomodulatory polypeptides can be effectively“loaded” onto the serum albumin, allowing a greater amount ofimmunomodulatory polypeptide to be successfully delivered to a site ofinflammation. The chemotherapeutic agent can be, for example, ananti-cancer chemotherapeutic agent. Such chemotherapeutic agentsinclude, but are not limited to, Gemcitabine, Docetaxel, Bleomycin,Erlotinib, Gefitinib, Lapatinib, Imatinib, Dasatinib, Nilotinib,Bosutinib, Crizotinib, Ceritinib, Trametinib, Bevacizumab, Sunitinib,Sorafenib, Trastuzumab, Ado-trastuzumab emtansine, Rituximab,Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate,Doxorubicin, Abraxane, Folfirinox, Cisplatin, Carboplatin,5-fluorouracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, andPemetrexed.

In some instances of the subject pharmaceutical compositions, thecomposition includes an immunomodulatory polypeptide that is aCD206-binding peptide (e.g., as described herein) and a chemotherapeuticagent. In some embodiments, the immunomodulatory polypeptide that findsuse in a combination composition is a peptide of Table 3. In certaininstances, the immunomodulatory peptide (e.g., a peptide of Table 3) iscombined with a chemotherapeutic agent. In certain cases of thepharmaceutical composition, the chemotherapeutic agent is Gemcitabine.In some cases of the pharmaceutical composition, the chemotherapeuticagent is Docetaxel. In some cases of the pharmaceutical composition, thechemotherapeutic agent is Abraxane.

In some instances of the subject pharmaceutical compositions, thecomposition includes an immunomodulatory polypeptide that is aCD206-binding peptide (e.g., as described herein) and is conjugated to asecond additional agent (e.g., as described herein). In some cases, theadditional agent is a chemotherapeutic agent. In some embodiments, theimmunomodulatory polypeptide that finds use in the subject peptideconjugate is a peptide of Table 3. In certain instances, theimmunomodulatory peptide (e.g., a peptide of Table 3) is conjugated to achemotherapeutic agent. In certain cases of the subject peptideconjugates, the chemotherapeutic agent is Gemcitabine. In some cases ofthe subject peptide conjugates, the chemotherapeutic agent is Docetaxel.In some cases of the subject peptide conjugates, the chemotherapeuticagent is Abraxane. In some cases of the subject peptide conjugates, thechemotherapeutic agent is paclitaxel.

In some cases, a subject pharmaceutical composition that finds use inthe treatment of cancer, e.g., ovarian cancer, includes animmunomodulatory polypeptide in combination with a vaccination therapy,e.g., a dendritic cell (DC) vaccination agent that promotes Th1/Th17immunity. In some cases of the pharmaceutical composition, theimmunomodulatory polypeptide is an adjuvant in combination with aTh17-inducing vaccination agent.

The pharmaceutical compositions of the present invention can beformulated for oral administration, parenteral administration,inhalation administration, topical administration, mucosaladministration, or the like. In some embodiments, the administering isvia a route selected from peroral, intravenous, intraperitoneal,inhalation, intranasal, intraprostatic, and intratumoral. The presentinvention is not limited by the route of administration. Compositionsformulated for oral delivery can, for example, include an enteric coat,to ensure that peptides contained therein reach the intestine andbeyond. Enteric formulations such as gastro resistant capsules for oraladministration, suppositories for rectal or vaginal administration alsoform part of this disclosure. Compositions formulated for topicaldelivery can be, for example, suspended in a gel or cream, coated on amicroneedle, or infused into a bandage or topical patch, to extend theduration of action of the peptides contained therein. Any inhalableformulation which can provide for an aerosolized form including asubject peptide for delivery to a patient via the intrapulmonary routemay be used in conjunction with the present disclosure. In some cases,the subject compositions are administered by intratumoral injection,e.g., into injectable cutaneous, subcutaneous, and/or nodal tumors.

In some embodiments, compositions are administered mucosally (e.g.,using standard techniques; See, e.g., Remington: The Science andPractice of Pharmacy, Mack Publishing Company, Easton, Pa., 19thedition, 1995 (e.g., for mucosal delivery techniques, includingintranasal, pulmonary, vaginal, and rectal techniques), as well asEuropean Publication No. 517,565 and Illum et al., J. Controlled Rel.,1994, 29:133-141 (e.g., for techniques of intranasal administration). Insome cases, the compositions of the present invention may beadministered dermally or transdermally using standard techniques.Methods of intranasal vaccination include the administration of adroplet or spray form of the subject composition into the nasopharynx ofa subject to be treated. In some embodiments, a nebulized or aerosolizedcomposition is provided.

Also provided are liposomal pharmaceutical compositions comprising thesubject immunomodulatory peptides. Any convenient nanocarriers andliposomes can be adapted for use in preparing liposomal formulations ofthe subject peptides, such as those nanocarriers and liposomes describedby Arias in “Liposomes in drug delivery: a patent review”. ExpertOpinion on Therapeutic Patents, 23, 2013, issue 11, p. 1399-1414; andTorchilin in “Multifunctional nanocarriers”, Advanced Drug DeliveryReviews, Volume 58, Issue 14, 1 December 2006, Pages 1532-1555.

Also provided are nanoparticle formulations or compositions includingthe subject immunomodulatory peptides. Nanopaticle formulations orcompositions can increase the aqueous solubility of a peptide ofinterest and can achieve protected, sustained, and targeted delivery ofthe peptide in therapeutic applications (e.g., as described herein). Insome cases, the formulation is a polymer-based nanoparticle formulation.Nanopaticle formulations of interest include albumin nanoparticles,e.g., human serum albumin containing nanoparticle formualtions. In somecases, a desolvation technique can be used for the preparation ofalbumin nanoparticles. Particle size, peptide drug release,encapsulation efficiency and peptide drug polymer interactions can bedetermined and selected using any convenient in vitro methods. Cellculture studies, in vivo pharmacokinetics, e.g., in rats, can be usedfor biological characterization of a desirable formulation.

In some instances, the nanoparticle formulation compositions includingthe subject immunomodulatory peptides is composed of iron oxidenanoparticles (IONPs). IONPs find use in a variety of biomedicalapplications. In some cases, an IONP formulation can exhibit high uptakein macrophages and/or target cancer cells. IONPs having a desirablecytotoxicity, in vivo distribution, and/or clearance can be selected foruse in conjunction with the subject immunomodulatory peptides. A varietyof well-characterized IONPs with different sizes and coatings can beutilized in the subject compositions and formulations. In some cases,polyethylenimine (PEI)-coated IONPs or PEGylated IONPs are utilized.IONPs can enhance cytotoxicity of the subject formulation throughmultiple mechanisms such as ROS production and apoptosis.

Also provided are kits including an immunomodulatory polypeptide that isa CD206-binding peptide (e.g., as described herein) and an additionalagent (e.g., a chemotherapeutic agent or an immunotherapeutic agent) foruse in treating cancer. The kit can include a dose of animmunomodulatory peptide in an amount effective to inhibit proliferationof cancer cells in a subject. The kit can also include a dose of anadditional agent, such as a chemotherapeutic agent or animmunotherapeutic agent (e.g., as described herein) in an amounteffective to inhibit proliferation of cancer cells in a subject. The kitin some cases includes an insert with instructions for administration ofthe immunomodulatory peptide and/or the additional agent (e.g., achemotherapeutic agent or an immunotherapeutic agent). In someinstances, the set of instructions for the combination therapy mayrecommend (i) a lower dose of the immunomodulatory peptide, when used incombination with the chemotherapeutic agent, (ii) a lower dose of theadditional agent (e.g., a chemotherapeutic agent or an immunotherapeuticagent), when used in combination with the immunomodulatory peptide,and/or (iii) a different dosing regimen for one or both agent than wouldnormally be recommended.

Methods

This disclosure provides methods of modulating macrophage activity usingan immunomodulatory peptide (e.g., as described herein). In some casesof the method, the macrophage activity that is modulated is macrophagepolarization. The method can include contacting a macrophage with aCD206-binding agent that is a peptide of this disclosure to modulateactivity of the macrophage. In some cases, by modulating acitivty ismeant inhibition of macrophage activity. The subject method can providefor reduction of the viability of the macrophage, which viability can bedetermined using any convenient methods.

In certain embodiments, an immunomodulatory polypeptide of thisdisclosure can bind to human CD206 with an affinity of at least −650kcal/mol, and in certain embodiments at least −700, −750, −800, −850,−900, −925, −950, −975, −1000, −1025, −1050 kcal/mol, or greater. Therequisite binding affinity can correspond to a binding affinity that canbe detected in vitro or in vivo. Alternatively, the requisite bindingaffinity can correspond to a binding affinity that can be detected insilico, e.g., using the ClusPro™ algorithm.

The macrophage targeted using the subject method can be a M2 macrophageor a tumor associated macrophage (TAM). The macrophage that is targetedcan be in vitro or in vivo.

In certain embodiments, a peptide of this disclosure binds to two ormore targets (e.g., pro-inflammatory targets). In some embodiments, avariant polypeptide binds to three, four, five, or more pro-inflammatorytargets. For example, a variant polypeptide can bind to any combinationof targets disclosed herein (e.g., an NF-kB Class II protein and humanserum albumin (HSA)), as discussed below. Such binding can be based onin silico, in vitro, or in vivo data.

Exemplary RP peptides of interest can interact with various signalingmolecules associated with inflammation, including NF-kB Class II subunitRelB, TGFβ, Notch1, Wnt8R, TRAIL, IL6R, IL10R, EGFR, and CDK6, as wellas other membrane associated signaling molecules, including CD206, CD47and SIRP-α, translational modification protein transglutaminase 2(TGM2), and histone modification enzyme histone methyl transferase(HMT). In certain cases, the subject peptides are CD206-bindingpeptides. Upon folding of these protein targets to their normal3-dimensional conformations, an amphipathic cleft is often generatedthat has high affinity for the immune-modulating peptides hereindescribed.

Further details of the target signaling molecules to which the subjectimmunomodulatory peptides specifically bind are set forth inWO2016/061133 by Jaynes et al., the disclosure of which is incorporatedherein in its entirety.

An immunomodulatory polypeptide that finds use in the subject methodscan be based on its ability to bind to the mannose-binding site on CD206and/or interfere with or block the binding of SIRP-mannose to CD206. Forexample, the immunomodulatory polypeptide can bind to at least one aminoacid residue of CD206 selected from the group consisting of Glu-725,Tyr-729, Glu-733, Asn-747, and Asp-748, or the equivalent amino acidresidue(s) in a CD206 protein of another species. Alternatively, theimmunomodulatory polypeptide can bind to at least one amino acid residueof human CD206 selected from the group consisting of Phe-708, Thr-709,Trp-710, Pro-714. Glu-719, Asn-720, Trp-721, Ala-722, Glu-725, Tyr-729,Glu-733, Asn-747, Asp-748, Ser-1691, Cys-1693, Phe-1694, and Phe-1703,or the equivalent amino acid residue(s) in a CD206 protein of anotherspecies. In certain embodiments, the immunomodulatory polypeptide canbind to at least one amino acid residue of CD206 selected from the groupconsisting of Phe-708, Trp-710, Trp-721, Glu-725, Tyr-729, Glu-733, orthe equivalent amino acid residue(s) in a CD206 protein of anotherspecies.

In certain instances, an immunomodulatory polypeptide binds afibronectin (FBN) domain of CD206 and/or interfere with or block thebinding of collagens to CD206. In some cases, the immunomodulatorypolypeptide can specifically bind a fibronectin (FBN) domain of CD206.In some instances, a subject immunomodulatory polypeptide binds a C-typecarbohydrate recognition domain (CRD) domain of the CD206 to modulate(e.g., activate) the activity of CD206. In some cases, a subjectimmunomodulatory polypeptide binds a C-type carbohydrate recognitiondomain (CRD) domain of the CD206 to modulate (e.g., interfere with,block or inhibit) the activity of CD206. In certain cases, the CRDdomain to which a subject immunomodulatory polypeptide specificallybinds to modulate the activity of CD206 is a CRD 4 or 5 domain.

In certain embodiments, an immunomodulatory polypeptide binds to two ormore targets (e.g., pro-inflammatory targets). In some embodiments, animmunomodulatory polypeptide binds to three, four, five, or morepro-inflammatory targets. For example, an immunomodulatory polypeptidecan bind to any combination of targets disclosed herein. Such bindingcan be based on in silico, in vitro, or in vivo data. Thus, animmunomodulatory polypeptide can bind to two or more NF-kB Class IIsubunits (e.g., RelB and at least one other NF-kB Class II subunit, suchas RelA, cRel, NF-kB1, or NF-kB2). Alternatively (or in addition), animmunomodulatory polypeptide can bind to an NF-kB Class II subunit(e.g., RelB) and at least one other signaling molecule (e.g., at leastone signaling molecule selected from the group consisting of TGFβ,Notch1, Wnt8R, TRAIL, IL6R, IL10R, EGFR, CDK6, CD206, CD47, SIRP-α, HMT,and TGM2). For example, an immunomodulatory polypeptide can bind to anNF-kB Class II subunit (e.g., RelB) and at least one signaling moleculeselected from the group consisting of TGFβ, Notch1, Wnt8R, TRAIL, IL6R,IL10R, EGFR, and CDK6. Alternatively, an immunomodulatory polypeptidecan bind to an NF-kB Class II subunit (e.g., RelB) and at least onesignaling molecule selected from the group consisting of CD206, CD47,SIRP-α, and TGM2. In other alternatives, an immunomodulatory polypeptidecan bind to an NF-kB Class II subunit (e.g., RelB) and HMT. In otheralternatives, an immunomodulatory polypeptide can bind to at least onesignaling molecule selected from the group consisting of TGFβ, Notch1,Wnt8R, TRAIL, IL6R, IL10R, EGFR, and CDK6, and at least one signalingmolecule selected from the group consisting of CD206, CD47, SIRP-α, andTGM2. In other alternatives, an immunomodulatory polypeptide can bind toat least one signaling molecule selected from the group consisting ofTGFβ, Notch1, Wnt8R, TRAIL, IL6R, IL10R, EGFR, and CDK6, and also bindto HMT. In still other embodiments, an immunomodulatory polypeptide canbind to an NF-kB Class II subunit (e.g., RelB), at least one signalingmolecule selected from the group consisting of TGFβ, Notch1, Wnt8R,TRAIL, IL6R, IL10R, EGFR, and CDK6, at least one signaling moleculeselected from the group consisting of CD206, CD47, SIRP-α, and TGM2, andalso HMT. In certain embodiments, an immunomodulatory polypeptide bindsto two or more pro-inflammatory targets and also serum albumin (e.g.,human serum albumin).

The immunomodulatory polypeptides of the present disclosure providepowerful tools for reducing inflammation and/or treating conditionsassociated with excessive inflammation (whether acute or chronic). Asused herein, the terms “treat,” “treating,” and similar words shall meanstabilizing, reducing the symptoms of, preventing the occurrence of, orcuring a medical condition.

Accordingly, the present disclosure provides methods of reducing theexpression level and/or activity of at least one (e.g., 2, 3, 4, 5, ormore) pro-inflammatory cytokine(s) at a site of inflammation in asubject. The methods include administering an immunomodulatorypolypeptide of the present disclosure (or, for example, a pharmaceuticalcomposition comprising an immunomodulatory polypeptide) to the subject.The pro-inflammatory cytokine can be selected from the group consistingof NF-kB, TNFα, IL-1, IL-6, IL-8, IL-12, IL-17, IL-23, MCP-1, MMP-1, andMMP-9. The reduction can be a reduction of at least 10% (e.g., 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, or more) in the expression or activity ofthe cytokine.

The present disclosure also provides methods of inhibiting an increasein the expression level and/or activity of at least one (e.g., 2, 3, 4,5, or more) pro-inflammatory cytokine(s) at a potential site ofinflammation in a subject. The methods include administering animmunomodulatory polypeptide of the present disclosure (or, for example,a pharmaceutical composition comprising an immunomodulatory polypeptide)to the subject. The pro-inflammatory cytokine can be selected from thegroup consisting of NF-kB, TNFα, IL-1, IL-6, IL-8, IL-12, IL-17, IL-23,MCP-1, MMP-1, and MMP-9. The methods can inhibit increased cytokineexpression and/or activity by limiting such increases to no more than20% (e.g., 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, 1%, or less).

It is understood that modulation of the level and/or activity ofpro-inflammatory cytokine(s) at a site of inflammation in a subject can,in some cases, provide for downstream modulation of the activity of animmune cells of interest (e.g., .effector T cells, regulatory T cells(Treg), natural killer cells (NK cells), B cells, and the like) andregulation of a target immune or inflammatory response.

The present disclosure also provides a method of treating or preventinga condition associated with chronic inflammation. The conditionassociated with chronic inflammation can be irritable bowel disease,ulcerative colitis, colitis, Crohn's disease, idiopathic pulmonaryfibrosis, asthma, keratitis, arthritis, osteoarthritis, rheumatoidarthritis, auto-immune diseases, a feline or human immunodeficiencyvirus (FIV or HIV) infection, cancer, age-related inflammation and/orstem cell dysfunction (e.g., age-related increases in Nlrp3 expression,age-related elevation of SOCS3 in muscle stem cells, etc.),graft-versus-host disease (GVHD), keloids, scleroderma, obesity,diabetes, diabetic wounds, other chronic wounds, atherosclerosis,multiple sclerosis, Parkinson's disease, Alzheimer's disease, maculardegeneration, gout, gastric ulcers, gastritis, mucositis, toxoplasmosis,and chronic viral or microbial infections (e.g., such as chronicbacterial or protozoan infections). The methods includes administeringan immunomodulatory polypeptide of the present disclosure (or, forexample, a pharmaceutical composition comprising an immunomodulatorypolypeptide) to a subject suffering from or likely to develop thecondition.

The present disclosure also provides methods of treating or preventingfibrosis. The fibrosis can be, for example, pulmonary fibrosis, dermalfibrosis, hepatic fibrosis, renal fibrosis, or fibrosis caused byionizing radiation. The methods include administering animmunomodulatory polypeptide of the present disclosure (or, for example,a pharmaceutical composition comprising an immunomodulatory polypeptide)to a subject suffering from or likely to develop fibrosis.

The present disclosure also provides methods of treating cancer. Thecancer can be colon cancer, breast cancer, leukemia, lymphoma, ovariancancer, prostate cancer, liver cancer, lung cancer, testicular cancer,cervical cancer, bladder cancer, endometrial cancer, kidney cancer,melanoma, cancers of the thyroid or brain, or ophthalmic cancer. Themethods include administering an immunomodulatory polypeptide of thepresent disclosure (or, for example, a pharmaceutical compositioncomprising an immunomodulatory polypeptide) to a subject suffering fromcancer. The presently disclosed subject matter also provides methods fortreating a solid tumor cancer in a subject. In some embodiments, themethod comprising administering the subject a therapeutically effectiveamount of a compound as disclosed herein.

For any of the foregoing methods, the subject can be an animal, such asa domesticated animal (e.g., a horse, cow, pig, goat, sheep, rabbit,chicken, turkey, duck, etc.), a pet (e.g., a dog, cat, rabbit, hamster,gerbil, bird, fish, etc.), a lab animal (e.g., a mouse, rat, monkey,chimpanzee, owl, fish, etc.), a zoo animal (e.g., a gorilla, orangutan,chimpanzee, monkey, elephant, camel, zebra, boar, lion, tiger, giraffe,bear, bird, etc.), a wild animal (e.g., a deer, wolf, mountain lion,bird, etc.), or a human.

In conjunction with any of the foregoing methods, the immunomodulatorypolypeptide(s) can be administered at a dose and frequency that dependson the type of animal, the size of the animal, and the condition beingtreated. Typically, the immunomodulatory polypeptide is administereddaily (or every other day, or weekly), in an amount between about 1 mgand about 1000 mg (e.g., about 5 mg to about 900 mg, about 5 mg to about800 mg, about 5 mg to about 700 mg, about 5 mg to about 600 mg, about 10mg to about 500 mg, about 10 mg to about 400 mg, about 10 mg to about300 mg, about 10 mg to about 250 mg, about 10 mg to about 200 mg, about10 mg to about 150 mg, about 10 mg to about 100 mg, about 50 mg to about500 mg, about 50 mg to about 400 mg, about 50 mg to about 300 mg, about50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about150 mg, about 50 mg to about 100 mg, about 75 mg to about 500 mg, about75 mg to about 400 mg, about 75 mg to about 300 mg, about 75 mg to about250 mg, about 75 mg to about 200 mg, about 75 mg to about 150 mg, about75 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg toabout 400 mg, about 100 mg to about 300 mg, about 100 mg to about 250mg, about 100 mg to about 200 mg, or any other range containing two ofthe foregoing endpoints). The daily dose can be administered once duringthe day, or broken up into smaller doses that are taken at multiple timepoints during the day. For a human (and other similarly-sized mammals),a dose of 5 mg/kg every other day can be administered. Theimmunomodulatory polypeptide can be administered for a fixed period oftime (e.g., for 2-3 weeks), at intervals (e.g., administer polypeptidefor 2-3 weeks, wait 2-3 weeks, then repeat the cycle), or until suchtime as the pro-inflammatory cytokine levels have been reduced orstabilized, the chronic inflammatory condition or fibrosis hasameliorated, or the cancer has gone into remission.

The administration of the immunomodulatory polypeptides (orpharmaceutical compositions comprising such polypeptides) in conjunctionwith any of the foregoing methods can be performed intravenously,intraperitoneally, parenteral, orthotopically, subcutaneously,topically, via inhalation, nasally, orally, sublingually, intraocularly,by means of an implantable depot, using nanoparticle-based deliverysystems, microneedle patch, microspheres, beads, osmotic or mechanicalpumps, and/or other mechanical means.

In conjunction with any of the foregoing methods, the immunomodulatorypolypeptides (e.g., as described herein) (or pharmaceutical compositionscomprising such polypeptides) can be administered in combination withanother drug designed to reduce or prevent inflammation, treat orprevent chronic inflammation or fibrosis, or treat cancer. In each case,the immunomodulatory polypeptide can be administered prior to, at thesame time as, or after the administration of the other drug. For thetreatment of cancer, the immunomodulatory polypeptide(s) can beadministered in combination with an additional therapeutic agent (e.g.,a chemotherapeutic agent or an immumotherapeutic agent) selected fromthe group consisting of taxanes, nucleoside analogs, steroids,anthracyclines, thyroid hormone replacement drugs, thymidylate-targeteddrugs, Chimeric Antigen Receptor/T cell therapies, Chimeric AntigenReceptor/NK cell therapies, apoptosis regulator inhibitors (e.g., B cellCLL/lymphoma 2 (BCL-2) BCL-2-like 1 (BCL-XL) inhibitors), CARP-1/CCAR1(Cell division cycle and apoptosis regulator 1) inhibitors,colony-stimulating factor-1 receptor (CSF1R) inhibitors, CD47inhibitors, cancer vaccine (e.g., a Th17-inducing dendritic cellvaccine) and other cell therapies. Specific chemotherapeutic agentsinclude, for example, Gemcitabine, Docetaxel, Bleomycin, Erlotinib,Gefitinib, Lapatinib, Imatinib, Dasatinib, Nilotinib, Bosutinib,Crizotinib, Ceritinib, Trametinib, Bevacizumab, Sunitinib, Sorafenib,Trastuzumab, Ado-trastuzumab emtansine, Rituximab, Ipilimumab,Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin,Abraxane, Folfirinox, Cisplatin, Carboplatin, 5-fluorouracil, Teysumo,Paclitaxel, Prednisone, Levothyroxine, Pemetrexed, navitoclax, ABT-199.

In some embodiments, the immunomodulatory polypeptide that finds use ina combination therapy is a peptide having macrophage modulating activity(e.g., as described herein). In certain instances, the immunomodulatorypolypeptide is a CD206-binding peptide (e.g., as described herein). Insome embodiments, the immunomodulatory polypeptide that finds use in acombination therapy is a peptide of Table 3. In certain instances, theimmunomodulatory peptide (e.g., a peptide of Table 3) can beadministered in combination with a chemotherapeutic agent to treatcancer. In certain cases, the chemotherapeutic agent is Gemcitabine. Insome cases, the chemotherapeutic agent is Docetaxel. In some cases, thechemotherapeutic agent is Abraxane.

For the treatment of cancer (e.g., melanoma, non-small cell lung canceror a lymphoma such as Hodgkin's lymphoma), the immunomodulatorypolypeptide(s) can be administered in combination with animmunotherapeutic agent. An immunotherapeutic agent is any convenientagent that finds use in the treatment of disease by inducing, enhancing,or suppressing an immune response. In some cases, the immunotherapeuticagent is an immune checkpoint inhibitor. Any convenient checkpointinhibitors can be utilized in combination with the subject peptides,including but not limited to, cytotoxic T-lymphocyte-associated antigen4 (CTLA-4) inhibitors, programmed death 1 (PD-1) inhibitors and PD-L1inhibitors. Exemplary checkpoint inhibitors of interest include, but arenot limited to, ipilimumab, pembrolizumab and nivolumab. In certainembodiments, for treatment of cancer and/or inflammatory disease, theimmunomodulatory polypeptide(s) can be administered in combination witha colony-stimulating factor-1 receptor (CSF1R) inhibitors. CSF1Rinhibitors of interest include, but are not limited to, emactuzumab.

Any convenient cancer vaccine therapies and agents can be used incombination with the subject immunomodulatory polypeptide compositionsand methods. For treatment of cancer, e.g., ovarian cancer, theimmunomodulatory polypeptide(s) can be administered in combination witha vaccination therapy, e.g., a dendritic cell (DC) vaccination agentthat promotes Th1/Th17 immunity. Th17 cell infiltration correlates withmarkedly prolonged overall survival among ovarian cancer patients. Insome cases, the immunomodulatory polypeptide finds use as adjuvanttreatment in combination with Th17-inducing vaccination.

Also of interest are agents that are CARP-1/CCAR1 (Cell division cycleand apoptosis regulator 1) inhibitors, including but not limited tothose described by Rishi et al., Journal of Biomedical Nanotechnology,Volume 11, Number 9, September 2015, pp. 1608-1627(20), and CD47inhibitors, including, but not limited to, anti-CD47 antibody agentssuch as Hu5F9-G4.

In certain instances, the combination provides an enhanced effectrelative to either component alone; in some cases, the combinationprovides a supra-additive or synergistic effect relative to the combinedor additive effects of the components. A variety of combinations of thesubject polypeptides and the chemotherapeutic agent may be employed,used either sequentially or simultaneously. For multiple dosages, thetwo agents may directly alternate, or two or more doses of one agent maybe alternated with a single dose of the other agent, for example.Simultaneous administration of both agents may also be alternated orotherwise interspersed with dosages of the individual agents. In somecases, the time between dosages may be for a period from about 1-6hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, toabout 1-2 week or longer following the initiation of treatment.

In some embodiments, the method is a method of reducing cancer cellproliferation, where the method includes contacting the cell with aneffective amount of a subject immunomodulatory polypeptide (e.g., asdescribed herein). The method can be performed in combination with achemotherapeutic agent (e.g., as described herein). The cancer cells canbe in vitro or in vivo. In certain instances, the method includescontacting the cell with an immunomodulatory peptide (e.g., a peptide ofTable 3) and contacting the cell with a chemotherapeutic agent. Anyconvenient cancer cells can be targeted. In certain cases, thechemotherapeutic agent is Gemcitabine. In some cases, thechemotherapeutic agent is Docetaxel. In some cases, the chemotherapeuticagent is Abraxane.

Alternatively, for the methods of treating cancer, the immunomodulatorypolypeptide(s) (or pharmaceutical compositions comprising suchpolypeptides) can be administered in combination with radiation therapy.Again, the immunomodulatory polypeptide(s) can be administered prior to,or after the administration of the radiation therapy.

Any of the foregoing methods of the present disclosure further include astep of assessing the efficacy of the therapeutic treatment. Because theimmunomodulatory polypeptides of the present disclosure have ademonstrable ability to reduce tissue inflammation and suppress theexcessive production of inflammatory mediators such as IL-1, IL-6,IL-12, and TNFα, both in tissues and in serum (data not shown), theefficacy of the therapeutic treatment can be assessed by measuring thelevels of such cytokines (e.g., in the serum) to determine whether thelevels have responded appropriately to the treatment. Depending on thecytokine levels, the dosage of immunomodulatory polypeptide(s) can beadjusted up or down, as needed.

Definitions

It is to be understood that this invention is not limited to particularembodiments described herein, which as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are described herein.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

EXAMPLES

Methods and materials of interest that find use in preparing andevaluating the subject immunomodulatory peptides include those disclosedin the experimental section of WO2016/061133 by Jaynes et al., thedisclosure of which is incorporated herein in its entirety.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent that the experiments below are all or the onlyexperiments performed.

Example 1 Suppression of Tumor Growth

The polypeptides of the present disclosure are also tested for theireffect on tumor growth in a mouse model of non-metastatic breast cancer.MCF-7 human non-metastatic breast cancer cells are cultured at 37° C.,5% CO2 in normal growth media. Cells are harvested at 80% to 90%confluence. Immune compromised athymic nude mice (J:NU) are divided into2 groups (9 animals per group). All mice are injected with ˜4.5×10⁶MCF-7 cells which had been stained with VIVO Tracker 680 and suspendedin 200 μl of PBS/Matrigel mixture. Cells are injected subcutaneously onthe dorsal surface of treated animals using a 22 gauge needle fittedwith a 500 μl syringe.

Animals are designated vehicle and peptide treated. The peptide treatedanimals are treated with the subject polypeptide. Freshly preparedpeptide s dissolved in sterile saline at a concentration of 100 μM andused to treat the animals in the peptide group. Vehicle treated animalsare injected with saline buffer alone. All treatments are injected intothe tumor mass two times weekly for 5 weeks using a 27½ gauge needlefitted with a 1 ml syringe Animal weights and tumor volumes are measured3 times weekly and the fluorescence labeling is followed by VIVO Tracker680 and IVIS Imaging.

FIG. 2 shows that peptides RP832C and RP185 reduce tumor volume in amouse tumor inhibition model. The data demonstrates that polypeptides ofthe present disclosure suppress tumor growth in vivo.

Example 2 Administering Peptides in Combination with Chemotherapy

Given the significant role of inflammation in tumor genesis andmetastasis, as well as the known association of M2 macrophage activitywith tumor development, it was anticipated that the administration ofpeptides of the present disclosure (e.g., selected peptides of Table 3)could positively influence the outcomes of cancer treatment.

To test this theory, cohorts of immunocompromised (“nude”) mice areinjected with ˜5×10⁶ human triple-negative breast cancer cells(MDA-MB-231) under the upper left teat. Following this administration,one cohort receives only vehicle; two of the cohorts receive thechemotherapeutic agent Gemcitabine, at a q4d dose of 40 mg/kg of bodyweight. One of these cohorts also received test peptide at a daily doseof 5 mg/kg body weight; and a fourth cohort received only peptide at adaily dose of 5 mg/kg body weight. Beginning on day 32 of the study, inthe Gemcitabine+RP-182 cohort, concentrations of RP-182 are increased to20 mg/kg body weight. Tumor volume is assessed at various time pointsfollowing initial cell administration. After 50 days, the mice aresacrificed.

In a second experiment, xenografts of C42B prostate cancer cells areintroduced into four cohorts of mice, and the tumors allowed to grow toapproximately 100 m³ before treatment. One cohort is treated only withvehicle; a second with Docetaxel at 2.5 mg/kg body weight administeredweekly; a third with test peptide administered daily subcu at 10 mg/kgbody weight; and a fourth with both Docetaxel at 2.5 mg/kg weekly andtest peptide at 10 mg/kg daily. Tumor volume is assessed at various timepoints following initial cell administration; after 27 days, the miceare sacrificed.

It is anticipated that the peptides of the present disclosure (e.g.,selected peptides of Table 3) will produce synergistic effects whenadministered with chemotherapeutic agents including Gemcitabine andDocetaxel, as well as checkpoint inhibitor therapies and otherimmunotherapies. In particular, the peptides of the present disclosuremay be particularly useful when used in conjunction withrecently-developed CAR-T (chimeric antigen receptor/T cell) therapies.Such therapies, while destroying tumor cells, create a very highsystemic burden of dead cell material, overstimulating the immune systemand creating a “cytokine storm” which can be fatal to the patient.

Example 3 Investigation of Selected Peptides

TABLE 5 The binding energies of selected peptides of Tables 3-4 forCD206 were calculated using the ClusPro algorithm: Binding Energy to RP#CD206 in ClusPro 832C −1312 837 −1218 182 −998 183 −943 107 −757 426−732 185 −953 186 −948 233 −713 851 −714 852 −963 853 −700

Example 4 Selective Effect of PP Peptides of Interest on SclerodermaMacrophage Viability

Peripheral blood derived macrophages were cultured from healthyvolunteers and scleroderma patients and cultured in MCS-F for 7 daysprior to qPCR for arginasel (M2 marker) or IFNg (M1 marker), followed bytreatment with RP peptides at a range of doses 0-100 uM. After 48 hoursmedia were exchanged and cells were retreated with the peptides. After96 hours cell viability was assayed by PrestoBlue assay. (FIG. 4A)Macrophages from a healthy control with low arginase:IFNg ratio of 3.6,were resistant to the effects of RP peptides of interest (RP182, RP185,RP832C, RP837) on viability. (FIG. 4B): In a scleroderma (SSc) patientwith a raised arginase:IFNg ratio of 8.8, RP peptides of interest(RP182, RP185, RP832C, RP837) even at 0.01 uM greatly reduced viabilityat 96 hours.

Example 5 Bleomycin Lung Fibrosis Rescue

Intratracheal instillation (IT) of bleomycin was used as a model forlung fibrosis. Rescue of bleomycin-induced lung fibrosis in mice by thesubject peptides was studied. Experimental parameters: Four groups (n=8)of C57BL6 male mice were studied over 6-8 weeks. 2.5 U/kg bleomycin wasadministered IT in a single bolus. After 72 hours, 1 mg/kg peptide ofinterest (RP182, RP185, RP832, RP837) was administered IT q2D. In thisbleomycin challenge experiment, the fibrosis measurements are Ashcroftscores following trichrome staining. Collagen scores are quantitativemeasurements following hydroxyproline staining. FIG. 1 shows a graph ofthese results.

The subject peptides reduced fibrosis and collagen deposition.Hematoxylin and eosin (H/E) and Mason's Trichrome staining of lungtissue sections was performed. The alveoli of the vehicle groups appearsurrounded by fibrotic tissue with increased collagen deposits unlikethe alveoli of the treated and naïve lung groups. Body weight changes:The body weight of RP peptide treatment groups showed no significantchange when compared to the body weight of the vehicle treated groups.Ashcroft score analysis: The vehicle group lung specimens showeddeformed lung architecture with increased fibrosis and collagendeposition and hence were assigned a score close to 6, unlike thepeptide-treated group that showed well organized lung architecture andhence a lower score.

Fibrosis and Collagen deposition level assessment: There was asignificant decrease of fibrosis and collagen deposition in the treatedgroup compared to the vehicle group as measured by ImageJ software. Lungweight changes: The vehicle group had higher lung weight compared to thepeptide treated group due to decreased fibrosis and collagen contents ofthe peptide treated group. IHC staining of TGFβ1 and αSMA: Peptidetreated lung tissues showed significant decrease in fibrosis associatedmarkers of TGFβ1 and αSMA.

These results demonstrate the exemplary peptides provided a reduction inbleomycin-induced lung fibrosis in a mouse model for lung fibrosis.

Example 6 RP Peptides of Interest Synergize with a PD-1 CheckpointInhibitor in CT26 Xenograft

FIG. 2 shows the results of a study of the effects of peptides ofinterest with and without an anti-PD1 antibody on tumor volume in amouse tumor inhibition model. The peptides RP832C and RP185 wereadministered at 10 mg/kg qd. The anti-PD1 antibody was administeredintraperitoneally at 200 ug twice per week. The results provided in theExamples demonstrate the efficacy of the peptides of the invention.

Nothwithstanding the appended claims, the following clauses are providedto illustrate aspects of the present disclosure.

Clause 1. An immunomodulatory peptide of 5 to 30 (e.g., 6 to 30 or 6 to18) amino acid residues in length, the peptide comprising: a striapathicregion of alternating hydrophilic and hydrophobic modules (e.g., havinga length of 6 to 12 or 6 to 10 amino acid residues) described by one offormulae 1-7 that adopts an amphipathic conformation under physiologicalconditions, comprising: 2 or more (e.g., 3 or more or 4 or more)hydrophobic modules; and one or more (e.g., 2 or more or 3 or more)hydrophilic modules each comprising at least one cationic residue;wherein the immunomodulatory peptide specifically binds CD206.

Clause 2. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 5 and has a sequence defined by theformula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]-[J_(3a)]-[X_(3a)]  (Formula5A);wherein: J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are eachindependently selected from a hydrophobic amino acid residue (e.g.,phenylalanine, tryptophan, alanine, valine, and glycine); and X_(1a),X_(1b), X_(2a), X_(2b) and X_(3a) are each independently selected from ahydrophilic amino acid residue (e.g., lysine, arginine, histidine,aspartic acid, glutamic acid, asparagine or glutamine).

Clause 3. The immunomodulatory peptide according to clause 2, wherein:J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are each independentlyselected from phenylalanine, tryptophan, alanine and glycine; andX_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are each independentlyselected from lysine and arginine.

Clause 4. The immunomodulatory peptide according to clause 2, wherein:J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are each independentlyselected from phenylalanine, tryptophan, alanine and valine; and X_(1a),X_(1b), X_(2a), X_(2b) and X_(3a) are each independently selectedornithine, lysine and arginine.

Clause 5. The immunomodulatory peptide according to one of clauses 2-4,having the sequence defined by the formula:FAX_(1a)X_(1b)FAX_(2a)X_(2b)J_(3a)FX_(3a) (SEQ ID NO: 30) whereinX_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are each independentlyselected from ornithine, lysine and arginine.

Clause 6. The immunomodulatory peptide according to clause 5, having thesequence FAOOFAOOFO (SEQ ID NO:19) (RP850).

Clause 7. The immunomodulatory peptide according to clause 2-4, havingthe sequence defined by the formula: FWKX_(1b)FVX_(2a)KWX_(3a) (SEQ IDNO: 31) wherein X_(1b), X_(2a) and X_(3a) are each independently lysineor arginine.

Clause 8. The immunomodulatory peptide according to clause 7, having asequence selected from FWKRFVRKWR (SEQ ID NO:4) (RP837) and FWKKFVKKWK(SEQ ID NO:7) (RP841).

Clause 9. The immunomodulatory peptide according to clause 8, wherein:J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are each tryptophan; andX_(1a), X_(1b), X_(2a), X_(2b) and X_(3a) are each independentlyselected from histidine, lysine and arginine.

Clause 10. The immunomodulatory peptide according to clause 9, havingthe sequence defined by the formula: WWX_(1a)HWWHX_(2b)WX_(3a) (SEQ IDNO: 32) wherein X_(1a), X_(2b) and X_(3a) are each independentlyhistidine, lysine or arginine.

Clause 11. The immunomodulatory peptide according to clause 10, having asequence selected from WWHHWWHHWH (SEQ ID NO:13), WWRHWWHRWR (SEQ IDNO:14), and WWKHWWHKWK (SEQ ID NO:15) (RP 847-849).

Clause 12. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 6 and has a sequence defined by theformula:[J_(1a)]-[X_(1a)X_(1b)]-[J_(2a)J₂]-[X_(2a)]-[J_(3a)]-[X_(3a)X_(3b)]-[J_(4a)J_(4b)]  (formula6A);

wherein J_(1a), J_(2a), J_(2b), J_(3a), J_(4a) and J_(4b) are eachindependently selected from a hydrophobic amino acid residue (e.g.,phenylalanine, tryptophan, alanine, isoleucine, valine, and glycine);and

X_(1a), X_(1b), X_(2a), X_(3a) and X_(3b) are each independentlyselected from a hydrophilic amino acid residue (e.g., lysine, arginine,histidine, aspartic acid, glutamic acid, asparagine or glutamine).

Clause 13. The immunomodulatory peptide according to clause 12, havingthe sequence GDRGIKGHRGF (SEQ ID NO:8) (RP842).

Clause 14. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 1 and has a sequence defined by oneof the formulae: [J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)] (Formula1A); and [J_(2b)J_(2a)]-[X_(1b)X_(1a)]-[J_(1b)J_(1a)] (Formula 1B);

wherein: J_(1a), J_(1b), J_(2a) and J_(2b) are each independentlyselected from a hydrophobic amino acid residue (e.g., phenylalanine,tryptophan and valine); and X_(1a) and X_(1b) are each independentlyselected from a hydrophilic amino acid residue (e.g., lysine orarginine).

Clause 15. The immunomodulatory peptide according to clause 14, having asequence FWKRFV (SEQ ID NO:5) (RP837N).

Clause 16. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 2 and has a sequence defined by theformula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)]-[X_(2a)]  (Formula 2A)wherein: J_(1a), J_(1b), J_(2a) and J_(2b) are each independentlyselected from a hydrophobic amino acid residue (e.g., phenylalanine,tryptophan and valine); and X_(1a), X_(1b) and X_(2a) are eachindependently selected from a hydrophilic amino acid residue (e.g.,lysine or arginine).

Clause 17. The immunomodulatory peptide according to clause 16, having asequence FVRKWR (SEQ ID NO:6) (RP837C¹).

Clause 18. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 4 and has a sequence defined by oneof the formulae:

[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]-[J_(3a)J_(3b)](Formula 4A); and[J_(3a)J_(3b)]-[X_(2a)X_(2b)]-[J_(2b)J_(2a)]-[X_(1b)X_(1a)]-[J_(1b)J_(1a)](Formula 4B); wherein J_(1a), J_(1b), J_(2a), J_(2b), J_(3a) and J_(3b)are each independently selected from a hydrophobic amino acid residue(e.g., phenylalanine, tyrosine or leucine); and X_(1a), X_(1b), X_(2a)and X_(2b) are each independently selected from a hydrophilic amino acidresidue (e.g., lysine or arginine).

Clause 19. The immunomodulatory peptide according to clause 18, havingthe sequence LYKKIIKKLL (SEQ ID NO:12) (RP846).

Clause 20. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 4 and has a sequence defined by theformula:[J_(1a)J_(1b)J_(1c)]-[X_(1a)]-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]-[J_(3a)J_(3b)]  (Formula4C);

wherein J_(1a), J_(1b), J_(1c), J_(2a), J_(2b), J_(3a), and J_(3b) areeach independently selected from a hydrophobic amino acid residue (e.g.,phenylalanine, tyrosine or proline); and X_(1a), X_(2a) and X_(2b) areeach independently selected from a hydrophilic amino acid residue (e.g.,aspartic acid, lysine or arginine).

Clause 21. The immunomodulatory peptide according to clause 20, havingthe sequence FYPDFFKKFF (SEQ ID NO:10) (RP844).

Clause 22. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 4 and has a sequence defined by theformula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)]-[X_(2a)X_(2b)X_(2c)]-[J_(3a)J_(3b)](Formula 4D); wherein J_(1a), J_(2a), J_(3a) and J_(3b) are eachindependently selected from a hydrophobic amino acid residue (e.g.,phenylalanine, serine, glycine or isoleucine); and X_(1a), X_(1b),X_(2a), X_(2b) and X_(2c) are each independently selected from ahydrophilic amino acid residue (e.g., glutamic acid, aspartic acid,lysine or arginine).

Clause 23. The immunomodulatory peptide according to clause 22, havingthe sequence FFRKSKEKIG (SEQ ID NO:18) (RP853).

Clause 24. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 4 and has a sequence defined by theformula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)]-[X_(2a)X_(2b)]-[J_(3a)](Formula 4E); wherein J_(1a), J_(2a), J_(2b), J_(2c), J_(3a), and J_(3b)are each independently selected from a hydrophobic amino acid residue(e.g., phenylalanine, alanine or isoleucine); and X_(1a), X_(1b),X_(2a), X_(2b) and X_(2c) are each independently selected from ahydrophilic amino acid residue (e.g., ornithine, lysine or arginine).

Clause 25. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 5 and has a sequence defined by theformula:[J_(1a)]-[X_(1a)]-[J_(2a)J_(2b)J_(2c)]-[X_(2a)]-[J_(3a)J_(3b)]-[X_(3a)X_(3b)](Formula 5C); wherein J_(1a), J_(2a), J_(2b), J_(2c), J_(3a), and J_(3b)are each independently selected from a hydrophobic amino acid residue(e.g., phenylalanine, leucine, glycine or isoleucine); and X_(1a),X_(2a), X_(3a) and X_(3b) are each independently selected from ahydrophilic amino acid residue (e.g., glutamine, lysine or histidine).

Clause 26. The immunomodulatory peptide according to clause 25, havingthe sequence FQFLGKIIHH (SEQ ID NO: 17) (RP852).

Clause 27. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 7 and has a sequence defined by theformula:[X_(1a)X_(1b)]-[J_(1a)]-[X_(2a)]-[J_(2a)]-[X_(3a)]-[J_(3a)J_(3b)J_(3c)](Formula 7A); wherein J_(1a), J_(2a), J_(3a), J_(3b), and J_(3c) areeach independently selected from a hydrophobic amino acid residue (e.g.,isoleucine, valine, leucine, serine or alanine); and X_(1a), X_(1b),X_(2a) and X_(3a) are each independently selected from a hydrophilicamino acid residue (e.g., lysine or arginine).

Clause 28. The immunomodulatory peptide according to clause 27, havingthe sequence KKIRVRLSA (SEQ ID NO: 16) (RP851).

Clause 29. The immunomodulatory peptide according to clause 1, whereinthe striapathic region is of Formula 5 and has a sequence defined by theformula:[J_(1a)J_(1b)]-[X_(1a)X_(1b)]-[J_(2a)J_(2b)J_(2c)]-[X_(2b)]-[J_(3a)]-[X_(3a)](Formula 5B);

wherein J_(1a), J_(1b), J_(2a), J_(2b) and J_(3a) are each independentlyselected from a hydrophobic amino acid residue (e.g., phenylalanine,alanine, threonine or leucine); and X_(1a), X_(1b), X_(2a), X_(2b) andX_(3a) are each independently selected from a hydrophilic amino acidresidue (e.g., histidine, aspartic acid, lysine or arginine).

Clause 30. The immunomodulatory peptide according to clause 29, havingthe sequence FFRHFATHLD (SEQ ID NO:11) (RP845).

Clause 31. The immunomodulatory peptide according to clause 40, whereinthe striapathic region is of Formula 3 and has a sequence defined by theformula:[X_(1a)X_(1b)]-[J_(1a)J_(1b)J_(1c)J_(1d)]-[X_(2a)X_(2b)]-[J_(2a)J_(2b)](Formula 3A); wherein: J_(1a), J_(1b), J_(1c), J_(1d), J_(2a) and J_(2b)are each independently selected from a hydrophobic amino acid residue(e.g., leucine, serine, alanine or phenylalanine); and X_(1a), X_(1b),X_(2a) and X_(2b) are each independently selected from a hydrophilicamino acid residue (e.g., glutamic acid, aspartic acid, lysine,asparagine or arginine).

Clause 32. The immunomodulatory peptide according to clause 31, having asequence EKLSAFRNFF (SEQ ID NO:9) (RP843).

Clause 33. The immunomodulatory peptide according to clause 1, whereinthe striapathic region comprises a dimer of first and secondpolypeptides connected via a peptide linker that connects the C-terminusof the first polypeptide and the N-terminus of the second polypeptide.

Clause 34. The immunomodulatory peptide according to clause 33, wherein:the hydrophilic modules consist of amino acid residues selected fromlysine, arginine and ornithine; and the hydrophobic modules consist ofamino acid residues selected from phenylalanine and tryptophan.

Clause 35. The immunomodulatory peptide according to clause 33 or 34,wherein the first and second polypeptides comprise one of the followingformulae: [X1]-[J1]-[X2]-[J2] (Formula 3); or [J1]-[X1]-[J2]-[X2](Formula 2).

Clause 36. The immunomodulatory peptide according to any one of clauses33-35, wherein the dimer has one of the following formulae:[X1]-[J1]-[X2]-[J2]-T-[J2]-[X2]-[J1]-[X1]; or[J1]-[X1]-[J2]-[X2]-T-[X2]-[J2]-[X1]-[J1]; wherein T is the peptidelinker.

Clause 37. The immunomodulatory peptide according to clause 36, whereinthe dimer has one of the following formulae:[X₁]-[J_(1a)]-[X_(2a)]-[J_(2a)]-T-[J_(2a)]-[X_(2a)]-[J_(1a)]-[X_(1a)](Formula 12A); or[J_(1a)]-[X_(1a)]-[J_(2a)]-[X_(2a)]-T-[X_(2a)]-[J_(2a)]-[X_(1a)]-[J_(1a)](Formula 13A); wherein T is the peptide linker (e.g., a polyglycinelinker).

Clause 38. The immunomodulatory peptide according to clause 37, having asequence selected from RWKFGGFKWR (SEQ ID NO:1) (RP832C) and FKWRGGRWKF(SEQ ID NO:3) (RP837C).

Clause 39. The immunomodulatory peptide according to clause 33 or 34,wherein the dimer has one of the following formulae:[X_(1a)X_(1b)]-[J_(1a)J_(1b)]-T-[J_(1b)J_(1a)]-[X_(1b)X_(1a)] (Formula8A); or [J_(1a)J_(1b)]-[X_(1a)X_(1b)]-T-[X_(1b)X_(1a)]-[J_(1b)J_(1a)](Formula 9A); wherein: T is the peptide linker (e.g., polyglycinelinker); J_(1a) and J_(1b) are each independently selected from ahydrophobic amino acid residue (e.g. tryptophan or phenylalanine); andX_(1a) and X_(1b) are each independently selected from a hydrophilicamino acid residue (e.g., asparagine or arginine).

Clause 40. The immunomodulatory peptide according to clause 49, havingthe sequence FWKRGGRKWF (SEQ ID NO:4) (peptide 837A).

Clause 41. The immunomodulatory peptide according to any one of clauses1-40, comprising:

-   -   a) a sequence selected from the peptide sequences of Table 3;    -   b) a sequence having at least 75% sequence identity (e.g., at        least 80%, at least 85%, at least 90% or at least 95% sequence        identity) with the sequence defined in a); or    -   c) a sequence having one or two amino acid substitutions        relative to the sequence defined in a), wherein the one or two        amino acid substitutions are substitutions for amino acids        according to Table 2 (e.g., a similar amino acid substitution, a        conservative amino acid substitutions or a highly conservative        amino acid substitution).

Clause 42. The immunomodulatory peptide according to clauses 1-41,consisting of a sequence selected from any one of the sequences of Table3 (SEQ ID NOs:1-19).

Clause 43. An immunomodulatory peptide of 6 to 30 amino acid residues inlength, comprising:

-   -   a) a peptide sequence selected from SEQ ID NO: (1-19) (e.g.,        RP832C, 837, 837A, 837C, 837N, 841-842, 843-850 and 853); or    -   b) a sequence having one or two amino acid substitutions        relative to the sequence defined in a), wherein the one or two        amino acid substitutions are substitutions for amino acids        according to Table 2 (e.g., a similar amino acid substitution, a        conservative amino acid substitutions or a highly conservative        amino acid substitution).

Clause 44. The immunomodulatory peptide of clause 43, wherein the one ortwo amino acid substitutions defined in b) consist of substitution of acationic amino acid of the sequence with an alternative cationic aminoacid residue (e.g., K for O, O for K, K for R, etc).

Clause 45. The immunomodulatory peptide of clause 43, comprising thepeptide sequence selected from: RWKFGGFKWR (RP832C) (SEQ ID NO: 1),FKWRGGRWKF (RP837C) (SEQ ID NO: 3) and FWKRGGRKWF (RP837A) (SEQ ID NO:4).

Clause 46. The immunomodulatory peptide of clause 43, comprising thepeptide sequence selected from FWKRFV (RP837N) (SEQ ID NO: 5) and FVRKWR(RP837C¹) (SEQ ID NO: 6).

Clause 47. The immunomodulatory peptide of clause 43, comprising apeptide sequence selected from FAOOFAOOFO (RP850) (SEQ ID NO: 19),FWKRFVRKWR (RP837) (SEQ ID NO: 4) and FWKKFVKKWK and (RP841) (SEQ ID NO:7).

Clause 48. The immunomodulatory peptide of clause 43, comprising apeptide sequence selected from WWHHWWHHWH (SEQ ID NO: 13), WWRHWWHRWR(SEQ ID NO: 14) and WWKHWWHKWK (SEQ ID NO: 15) (RP847-849).

Clause 49. The immunomodulatory peptide of clause 43, comprising thepeptide sequence GDRGIKGHRGF (RP842) (SEQ ID NO: 8).

Clause 50. The immunomodulatory peptide of clause 43, comprising thepeptide sequence LYKKIIKKLL (RP846) (SEQ ID NO: 12).

Clause 51. The immunomodulatory peptide of clause 43, comprising thepeptide sequence FYPDFFKKFF (RP844) (SEQ ID NO: 10).

Clause 52. The immunomodulatory peptide of clause 43, comprising thepeptide sequence FFRKSKEKIG (RP853) (SEQ ID NO: 18).

Clause 53. The immunomodulatory peptide of clause 43, comprising thepeptide sequence FFRHFATHLD (RP845) (SEQ ID NO: 11).

Clause 54. The immunomodulatory peptide of clause 43, comprising thepeptide sequence EKLSAFRNFF (RP843) (SEQ ID NO: 9).

Clause 55. An immunomodulatory peptide (e.g., of 12 amino acid residuesor less in length), comprising a sequence selected from: RWKFGGFKWR(RP832C) (SEQ ID NO: 1), FKWRGGRWKF (RP837C) (SEQ ID NO: 3) andFWKRGGRKWF (RP837A) (SEQ ID NO: 4).

Clause 56. The immunomodulatory peptide of clause 55, consisting of thesequence: RWKFGGFKWR (RP832C) (SEQ ID NO: 1).

Clause 57. The immunomodulatory peptide of clause 55, consisting of thesequence: FKWRGGRWKF (RP837C) (SEQ ID NO: 3).

Clause 58. The immunomodulatory peptide of clause 55, consisting of thesequence: FWKRGGRKWF (RP837A) (SEQ ID NO: 4).

Clause 59. A pharmaceutical composition, comprising the immunomodulatorypeptide of any one of clauses 1-58 and a pharmaceutically acceptablecarrier.

Clause 60. The pharmaceutical composition of clause 59, wherein thecomposition is formulated for oral administration, parenteraladministration, administration via inhalation, or topicaladministration.

Clause 61. The pharmaceutical composition of clause 59 or 60, whereinthe composition is formulated for intravenus or subcutaneousadministration.

Clause 62. The pharmaceutical composition of clause 59 or 60, whereinthe composition is formulated for oral administration and furthercomprises an enteric coating.

Clause 63. The pharmaceutical composition of clause 59 or 60, whereinthe composition is formulated for topical delivery in a form selectedfrom the group consisting of: a gel suspension, a cream, microneedle,and infused into a bandage or topical patch.

Clause 64. A method of modulating macrophage activity, the methodcomprising: contacting a macrophage with a CD206-binding agent tomodulate activity of the macrophage.

Clause 65. The method according to clause 64, wherein the CD206-bindingagent binds to a mannose-binding site to modulate binding of signalregulatory protein (SIRP)-mannose to CD206.

Clause 66. The method according to any one of clauses 64-65, wherein theCD206-binding agent binds to CD206 with a binding energy of at least−650 kcal/mol.

Clause 67. The method according to any one of clauses 64-66, wherein theCD206-binding agent directly contacts at least one amino acid residue ofCD206 selected from Phe-708, Thr-709, Trp-710, Pro-714, Glu-719,Asn-720, Trp-721, Ala-722, Glu-725, Tyr-729, Glu-733, Asn-747, Asp-748,Ser-1691, Cys-1693, Phe-1694 and Phe-1703.

Clause 68. The method according to any one of clauses 64-67 wherein themacrophage activity that is modulated is macrophage polarization.

Clause 69. The method according to any one of clauses 64-68, whereinviability of the macrophage is reduced.

Clause 70. The method according to any one of clauses 64-69, wherein themacrophage is a M2 macrophage or a tumor associated macrophage (TAM).

Clause 71. The method according to any one of clauses 64-70, wherein theCD206-binding agent inhibits macrophage activity.

Clause 72. The method according to any one of clauses 64-71, wherein theCD206-binding agent is an immunomodulatory peptide.

Clause 73. The method according to any one of clauses 64-71, wherein themacrophage is in vitro.

Clause 74. The method according to any one of clauses 64-71, wherein themacrophage is in vivo.

Clause 75. The method according to any one of clauses 64-74, wherein theCD206-binding agent is an immunomodulatory peptide according to any oneof clauses 1-58.

Clause 76. A method of treating a subject for a condition associatedwith chronic inflammation, the method comprising: administering aneffective amount of a CD206-binding agent (e.g., an immunomodulatorypeptide according to any one of clauses 1-58) to the subject to treatthe subject for the condition associated with chronic inflammation.

Clause 77. The method according to clause 76, wherein the conditionassociated with chronic inflammation is selected from the groupconsisting of scleroderma or multiple sclerosis, irritable boweldisease, ulcerative colitis, colitis, Crohn's disease, idiopathicpulmonary fibrosis, scleroderma, asthma, keratitis, arthritis,osteoarthritis, rheumatoid arthritis, auto-immune diseases, a feline orhuman immunodeficiency virus (FIV or HIV) infection, cancer, age-relatedinflammation and/or stem cell dysfunction, graft-versus-host disease(GVHD), keloids, obesity, diabetes, diabetic wounds, other chronicwounds, atherosclerosis, Parkinson's disease, Alzheimer's disease,macular degeneration, gout, gastric ulcers, gastritis, mucositis,toxoplasmosis, and chronic viral or microbial infections.

Clause 78. The method according to any one of clauses 76-77, wherein theCD206-binding agent is an immunomodulatory peptide according to any oneof clauses 1-58.

Clause 79. The method according to clause 77, wherein the condition iscancer.

Clause 80. The method according to clause 79, further comprisingadministering an effective amount of an additional agent to the subject.

Clause 81. The method according to clause 80, where the additional agentis a chemotherapeutic agent.

Clause 82. The method according to clause 80, wherein thechemotherapeutic agent is selected from Gemcitabine, Docetaxel,Bleomycin, Erlotinib, Gefitinib , Lapatinib, Imatinib, Dasatinib,Nilotinib, Bosutinib, Crizotinib, Ceritinib, Trametinib, Bevacizumab,Sunitinib, Sorafenib, Trastuzumab, Ado-trastuzumab emtansine, Rituximab,Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate,Doxorubicin, Abraxane, Folfirinox, Cisplatin, Carboplatin,5-fluorouracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, andPemetrexed.

Clause 83. The method according to clause 81, wherein thechemotherapeutic agent is abraxane.

Clause 84. The method according to clause 81, wherein thechemotherapeutic agent is Gemcitabine or Docetaxel.

Clause 85. The method according to clause 80, where the additional agentis an immunotherapeutic agent.

Clause 86. The method according to clause 85, wherein theimmunotherapeutic agent is an immune checkpoint inhibitor.

Clause 87. The method according to clause 86, wherein the immunecheckpoint inhibitor is selected from cytotoxic T-lymphocyte-associatedantigen 4 (CTLA-4) inhibitors, programmed death 1 (PD-1) inhibitors andPD-L1 inhibitors.

Clause 88. The method according to clause 87, wherein the immunecheckpoint inhibitor is selected from ipilimumab, pembrolizumab andnivolumab.

Clause 89. The method according to clause 76, wherein the conditionassociated with chronic inflammation is a fibrosis.

Clause 90. The method according to clause 76, wherein the conditionassociated with chronic inflammation is scleroderma.

Clause 91. The method according to any one of clauses 76-90, wherein theCD206-binding agent is an immunomodulatory peptide according to any oneof clauses 1-58.

Clause 92. The method according to clause 91, wherein the CD206-bindingagent consists of an immunomodulatory peptide of Table 3.

What is claimed is:
 1. An immunomodulatory peptide, comprising: apeptide sequence selected from the group consisting of FAOOFAOOFO(RP850) (SEO ID NO: 19), FWKRFVRKWR (RP837) (SEO ID NO: 2) andFWKKFVKKWK (RP841) (SEO ID NO: 7).
 2. The immunomodulatory peptide ofclaim 1, comprising the peptide sequence FWKRFVRKWR (RP837) (SEQ ID NO:2).
 3. A pharmaceutical composition, comprising the immunomodulatorypeptide of claim 1 and a pharmaceutically acceptable carrier.
 4. Amethod of treating a subject for a condition associated with chronicinflammation, the method comprising: administering an effective amountof a peptide according to claim 1 to the subject to treat the subjectfor the condition associated with chronic inflammation, wherein thecondition associated with chronic inflammation is selected from thegroup consisting of scleroderma and lung fibrosis.
 5. The methodaccording to claim 4, wherein the condition associated with chronicinflammation is lung fibrosis.
 6. The method according to claim 4,wherein the condition associated with chronic inflammation isscleroderma.
 7. The method according to claim 4, wherein two or moredifferent peptides each according to claim 1 are administered to thesubject.
 8. An immunomodulatory peptide, consisting of: a peptidesequence selected from the group consisting of FWKRFVRKWR (RP837) (SEQID NO: 2), FWKKFVKKWK (RP841) (SEQ ID NO: 7), and FAOOFAOOFO (RP850)(SEQ ID NO:
 19. 9. The immunomodulatory peptide of claim 8, consistingof the peptide sequence FWKRFVRKWR (RP837) (SEQ ID NO: 2).
 10. Thepharmaceutical composition of claim 3, wherein the composition isformulated for oral administration, parenteral administration,administration via inhalation, or topical administration.
 11. Thepharmaceutical composition of claim 3, wherein the composition isformulated for intravenous or subcutaneous administration.
 12. Thepharmaceutical composition of claim 3, wherein the composition isformulated for intratumoral administration.
 13. The pharmaceuticalcomposition of claim 3, wherein the composition is formulated for oraladministration and further comprises an enteric coating.
 14. Thepharmaceutical composition of claim 3, wherein the composition isformulated for topical delivery in a form selected from: gel suspension,cream, microneedle and infused into a bandage or topical patch.
 15. Thepharmaceutical composition of claim 3, wherein the immunomodulatorypeptide comprises the sequence FAOOFAOOFO (RP850) (SEQ ID NO: 19). 16.The pharmaceutical composition of claim 3, wherein the immunomodulatorypeptide comprises the sequence FWKRFVRKWR (RP837) (SEQ ID NO: 2). 17.The pharmaceutical composition of claim 3, wherein the immunomodulatorypeptide comprises the sequence FWKKFVKKWK (RP841) (SEQ ID NO: 7).